Human aspartic proteases

ABSTRACT

The invention provides human aspartic proteases (NHAP) and polynucleotides which identify and encode NHAP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of NHAP.

[0001] This application is a divisional application of co-pending U.S. Ser. No. 09/705,448, entitled “Human Aspartic Proteases,” filed Nov. 2, 2000, which is a divisional application of U.S. Ser. No. 09/116, 641, filed Jul. 16, 1998, now abandoned, and which is a continuation-in-part of Ser. No. 09/008,271, filed on Jan. 16, 1998.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequences of aspartic proteases and to the use of these sequences in the diagnosis, treatment, and prevention of respiratory, endocrinological, and immunological disorders, and cancer.

BACKGROUND OF THE INVENTION

[0003] Proteolytic processing is an essential component of normal cell growth, differentiation, remodeling, and homeostasis. The cleavage of peptide bonds within cells is necessary for the maturation of precursor proteins to their active form, the removal of signal sequences from targeted proteins, the degradation of incorrectly folded proteins, and the controlled turnover of peptides within the cell. Proteases participate in apoptosis, inflammation, and in tissue remodeling during embryonic development, wound healing, and normal growth. They are necessary components of bacterial, parasitic, and viral invasion and replication within a host. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York, N.Y., pp. 1-5.)

[0004] One category is the cysteine proteases involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation of proteins. Cysteine proteases are produced by monocytes, macrophages and other cells of the immune system which migrate to sites of inflammation and in their protective role secrete various molecules to repair damaged tissue. These cells may overproduce the same molecules and cause tissue destruction in certain disorders. The cathepsin family of lysosomal proteases includes the cysteine proteases, including cathepsins B, H, K, L, O2, and S, and the aspartic proteases, including pepsin A, gastricsin, chymosin, renin, and cathepsins D and E. Various members of this endosomal protease family are differentially expressed. Some, such as cathepsin D, have a ubiquitous tissue distribution while others, such as cathepsin L, are found only in monocytes, macrophages, and other cells of the immune system.

[0005] The characteristic active site residues of aspartic proteases are a pair of aspartic acid residues, e.g., asp33 and asp213 in penicillopepsin. Aspartic proteases are also called acid proteases because the optimum pH for activity is between 2 and 3. In this pH range, one of the aspartate residues is ionized, the other un-ionized. A potent inhibitor of aspartic proteases is the hexapeptide, pepstatin, which in the transition state resembles the normal substrate.

[0006] Abnormal regulation and expression of cathepsins is evident in various inflammatory disease states. In autoimmune diseases such as rheumatoid arthritis, the secretion of the cysteine protease, cathepsin C, degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. In cells isolated from inflamed synovia, the mRNA for stromelysin, cytokines, TIMP-1, cathepsin, gelatinase, and other molecules is preferentially expressed. Expression of cathepsins L and D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium. (Keyszer, G. M. (1995) Arthritis Rheum. 38:976-984.) The increased expression and differential regulation of the cathepsins is linked to the metastatic potential of a variety of cancers and as such is of therapeutic and prognostic interest. (Chambers, A. F. et al. (1993) Crit. Rev. Oncog. 4:95-114.)

[0007] The discovery of new aspartic proteases and the polynucleotides encoding then satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of respiratory, endocrinological, and immunological disorders, and cancer.

SUMMARY OF THE INVENTION

[0008] The invention features substantially purified polypeptides, aspartic proteases, referred to collectively as “NHAP” and individually as “NHAP-1” and “NHAP-2.” In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0009] The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:3, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0010] Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0011] The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4. The invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.

[0012] The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. In another aspect, the expression vector is contained within a host cell.

[0013] The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.

[0014] The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.

[0015] The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as a purified agonist and a purified antagonist to the polypeptide.

[0016] The invention also provides a method for treating or preventing an endocrinological disorder associated with decreased expression or activity of NHAP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0017] The invention also provides a method for treating or preventing an endocrinological disorder associated with increased expression or activity of NHAP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3

[0018] The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0019] The invention also provides a method for treating or preventing an immunological disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0020] The invention also provides a method for treating or preventing a respiratory disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

[0021] The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0022]FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID NO:2) of NHAP-1. The alignment was produced using MacDNASIS PRO software (Hitachi Software Engineering Co. Ltd., San Bruno, Calif.).

[0023]FIGS. 2A, 2B, 2C, and 2D show the amino acid sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of NHAP-2. The alignment was produced using MacDNASIS PRO software

[0024]FIGS. 3A, 3B, and 3C show the amino acid sequence alignments among NHAP-1 (372637; SEQ ID NO:1), NHAP-2 (2435410; SEQ ID NO:3), and a mouse kidney-derived, aspartic protease-like protein (GI 1906810; SEQ ID NO:10), produced using the multisequence alignment program of LASERGENE software (DNASTAR Inc, Madison Wis.).

[0025]FIG. 4 shows the northern analysis of NHAP-1 and NHAP-2 probed with NHAP-2 cDNA. Tissue blots were obtained from Clontech, Palo Alto, Calif.

[0026]FIG. 5 shows the northern analysis of NHAP-1 probed with NHAP-1-specific oligonucleotide using the same tissue blots as in FIG. 4.

[0027]FIG. 6 shows western analysis of recombinant NHAP-1 protein expression in Escherichia coli. Competent E. Coli strain BL21 (DE3) was transformed with either vector (pET15b) or with NHAP-1 expression construct (pET15b/NHAP-1). Cell lysates from cultures before IPTG induction (P) or after IPTG induction (I) were separated using polyacrylamide gel electrophoresis under reduced denatured conditions, and probed with preimmune and immune serums (IC620).

[0028] Table 1 shows the Incyte clone and the associated library in which nucleic acid sequences encoding NHAP were identified, a brief description of the library, and the vector into which each cDNA was cloned.

[0029] Table 2 summarizes the databases and tools used to assemble and analyze the sequences of the invention. The first column of Table 2 shows the tool, program, or algorithm; the second column, the database; the third column, a brief description; and the fourth column (where applicable), scores for determining the strength of a match between two sequences (the higher the value, the more homologous).

DESCRIPTION OF THE INVENTION

[0030] Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0031] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

[0033] “NHAP,” as used herein, refers to the amino acid sequences, or variant thereof, of substantially purified NHAP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.

[0034] The term “agonist,” as used herein, refers to a molecule which, when bound to NHAP, increases or prolongs the duration of the effect of NHAP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of NHAP.

[0035] An “allelic variant,” as this term is used herein, is an alternative form of the gene encoding NHAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0036] “Altered” nucleic acid sequences encoding NHAP, as described herein, include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as NHAP or a polypeptide with at least one functional characteristic of NHAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding NHAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding NHAP. The encoded protein may also be “altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent NHAP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of NHAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.

[0037] The terms “amino acid” or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. In this context, “fragments,” “immunogenic fragments,” or “antigenic fragments” refer to fragments of NHAP which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of NHAP. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

[0038] “Amplification,” as used herein, relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., pp.1-5.)

[0039] The term “antagonist,” as it is used herein, refers to a molecule which, when bound to NHAP, decreases the amount or the duration of the effect of the biological or immunological activity of NHAP. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of NHAP.

[0040] As used herein, the term “antibody” refers to intact molecules as well as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind NHAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

[0041] The term “antigenic determinant,” as used herein, refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

[0042] The term “antisense,” as used herein, refers to any composition containing a nucleic acid sequence which is complementary to the “sense” strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.

[0043] As used herein, the term “biologically active,” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic NHAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

[0044] The terms “complementary” or “complementarity,” as used herein, refer to the natural binding of polynucleotides by base pairing. For example, the sequence “5′ A-G-T 3′” binds to the complementary sequence “3′ T-C-A 5′.” Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.

[0045] A “composition comprising a given polynucleotide sequence” or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding NHAP or fragments of NHAP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts, e.g., NaCl, detergents, e.g.,sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

[0046] “Consensus sequence,” as used herein, refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCR (Applied Biosystems (ABI), Foster City Calif.) in the 5′ and/or the 3′ direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW Fragment Assembly system (GCG, Madison, Wis.). Some sequences have been both extended and assembled to produce the consensus sequence.

[0047] As used herein, the term “correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding NHAP, by Northern analysis is indicative of the presence of nucleic acids encoding NHAP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding NHAP.

[0048] A “deletion,” as the term is used herein, refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

[0049] The term “derivative,” as used herein, refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

[0050] The term “similarity,” as used herein, refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.” A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

[0051] The phrases “percent identity” or “% identity” refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.

[0052] “Human artificial chromosomes” (HACs), as described herein, are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)

[0053] The term “humanized antibody,” as used herein, refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

[0054] “Hybridization,” as the term is used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

[0055] As used herein, the term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

[0056] The words “insertion” or “addition,” as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.

[0057] “Immune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.

[0058] The term “microarray,” as used herein, refers to an arrangement of distinct polynucleotides arrayed on a substrate, e.g., paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.

[0059] The terms “element” or “array element” as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.

[0060] The term “modulate,” as it appears herein, refers to a change in the activity of NHAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NHAP.

[0061] The phrases “nucleic acid” or “nucleic acid sequence,” as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In this context, “fragments” refers to those nucleic acid sequences which, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.

[0062] The terms “operably associated” or “operably linked,” as used herein, refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.

[0063] The term “oligonucleotide,” as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. As used herein, the term “oligonucleotide” is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.

[0064] “Peptide nucleic acid” (PNA), as used herein, refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63.)

[0065] The term “sample,” as used herein, is used in its broadest sense. A biological sample suspected of containing nucleic acids encoding NHAP, or fragments thereof, or NHAP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solid support; a tissue; a tissue print; etc.

[0066] As used herein, the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope “A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

[0067] As used herein, the term “stringent conditions” refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.

[0068] The term “substantially purified,” as used herein, refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.

[0069] A “substitution,” as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

[0070] “Transformation,” as defined herein, describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed” cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0071] A “variant” of NHAP polypeptides, as used herein, refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

[0072] The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to NHAP. This definition may also include, for example, “allelic” (as defined above), “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.

THE INVENTION

[0073] The invention is based on the discovery of two new human aspartic proteases (NHAP), the polynucleotides encoding NHAP, and the use of these compositions for the diagnosis, treatment, or prevention of respiratory, endocrinological, and immunological disorders, and cancer.

[0074] Nucleic acids encoding the NHAP-1 and NHAP-2 of the present invention were identified in the following Incyte Clones: (SEQ ID NO:5 through 9) 372637H1 (LUNGNOT02), 1242901H1 (LUNGNOT03), 2222291H1 (LUNGNOT18), 2435410H1 (EOSINOT03), and 2756549H1 (THP1AZS08) using a computer search, e.g., BLAST, for amino acid sequence alignments. The full length cDNA sequence of NHAP- 1 (SEQ ID NO:2) was obtained from a human lung cDNA library using the GENETRAPPER method (Invitrogen, Carlsbad Calif.) and oligonucleotides derived from Incyte clone 2756549 (THP1AZS08). The full length cDNA sequence of NHAP-2 (SEQ ID NO:4) was obtained from a human leukocyte cDNA library using the GENETRAPPER method (Invitrogen) and the same oligonucleotides as were used for NHAP-1.

[0075] In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C, and 1D. NHAP-1 is 420 amino acids in length and has a potential signal peptide sequence extending from residues M1 to P21. Potential N-glycosylation sites are found at residues N90, N133, and N336. Potential phosphorylation sites are found for casein kinase II at S60 and T338, and for protein kinase C at S106, T143, T346, and S393. Two potential leucine zipper patterns are found beginning at L309 and L316, and a potential cell attachment site is found in the sequence R387GD. Two potential active site aspartate residues, characteristic of aspartic proteases, are found at residues D96 and D283. BLOCKS and PRINTS analyses also identify sequences encompassing the two aspartate residues as characteristic of aspartic proteases. As shown in FIGS. 3A, 3B, and 3C, NHAP-1 has chemical and structural similarity with a mouse aspartic protease-like protein (GI 1906810; SEQ ID NO:10). In particular, NHAP-1 and the mouse aspartic protease-like protein share 69% identity. The two proteins share the signal sequence, the three potential glycosylation sites, and the potential phosphorylations sites found in NHAP-1 at S106, T143, and T338. The two potential active site aspartate residues found in NHAP-1 and NHAP-2, and the surrounding sequences, are also conserved in the mouse protein. The fragment of SEQ ID NO:2 from about nucleotide 160 to about nucleotide 228, which encodes a fragment of SEQ ID NO:1 from about amino acid residue P54 to about amino acid residue V76, is useful, for example, as a hybridization probe.

[0076] In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2A, 2B, 2C, and 2D. NHAP-2 is 433 amino acids in length and has a potential signal sequence extending from residues M1 to P21, three potential N-glycosylation sites at N90, N125, and N336, potential phosphorylation sites for cAMP-cGMP-dependent protein kinase at T413, for casein kinase at S60, S181, T338, and T383, for protein kinase C at S106, S129, and T143, and for tyrosine kinase at Y78, and a potential cell attachment site is found in the sequence R387GD. Two potential active site aspartate residues, characteristic of aspartic proteases, are found at residues D96 and D283. BLOCKS and PRINTS analyses also identify sequences encompassing the two aspartate residues as characteristic of aspartic proteases. As shown in FIGS. 3A, 3B, and 3C, NHAP-2 has chemical and structural similarity with a mouse aspartic protease-like protein (GI 1906810; SEQ ID NO:10). In particular, NHAP-2 and the mouse aspartic protease-like protein share 69% identity, the two potential glycosylation sites at N90 and N336, and the potential phosphorylations sites found in NHAP-2 at S106, S129, T143 and T338. The two potential active site aspartate residues found in NHAP-2, and their surrounding sequences, are also conserved in the mouse protein. The sequence of SEQ ID NO:4 from about nucleotide 190 to about nucleotide 258, which encodes a fragment of SEQ ID NO:3 from about amino acid residue P54 to about amino acid residue A76, is useful, for example, as a hybridization probe.

[0077] Electronic northern analysis shows clones clustered with NHAP expressed in a variety of cDNA libraries at least 59% of which involve cancer and immortalized cell lines, and at least 22% of which involve inflammation and the immune response. Of particular note is the expression of NHAP in lung tissue (37%). Membrane based northern analysis using NHAP-2 cDNA showed the expression of an ˜1.3 kb RNA species in kidney, lung, and tissues associated with the immune response, including spleen, bone marrow, and peripheral blood leukocytes (FIG. 4). Since the NHAP-2 probe has ˜90% homology to NHAP-1, the analysis represents the expression of both NHAP-1 and NHAP-2. Membrane based northern analysis using an oligonucleotide probe specific for NHAP-1 (FIG. 5) showed the expression of the ˜1.3 kb RNA species only in lung. Immunocytochemical staining of normal and diseased human tissue samples using NHAP-1 specific rabbit immune serum demonstrated the expression of the protein in pituitary gland, thyroid follicular cells, normal lung alveoli, bronchioloalveolar carcinoma and lung adenocarcinoma.

[0078]FIG. 6 shows the western analysis of recombinant NHAP-1 protein expressed in E. Coli. NHAP-1 was detected as a band of around 45 kDa using immune, but not preimmune, serum and was found predominantly in IPTG-induced cells containing the NHAP- 1 expression construct.

[0079] Chromosomal localization studies by FISH analysis revealed that genes encoding NHAP-1 and NHAP-2 were localized to the long arms of chromosome 19, specifically to an area corresponding to band 19q13.3.

[0080] The invention also encompasses NHAP variants. A preferred NHAP variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the NHAP amino acid sequence, and which contains at least one functional or structural characteristic of NHAP.

[0081] The invention also encompasses polynucleotides which encode NHAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO:2, which encodes an NHAP. In a further embodiment, the invention encompasses the polynucleotide sequence comprising the sequence of SEQ ID NO:4, which encodes an NHAP.

[0082] The invention also encompasses a variant of a polynucleotide sequence encoding NHAP. In particular, such a variant polynucleotide sequence will have at least about 70%, more preferably at least about 80%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding NHAP. A particular aspect of the invention encompasses a variant of SEQ ID NO:2 which has at least about 70%, more preferably at least about 80%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:2. The invention further encompasses a polynucleotide variant of SEQ ID NO:4 having at least about 70%, more preferably at least about 80%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:4. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of NHAP.

[0083] It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding NHAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring NHAP, and all such variations are to be considered as being specifically disclosed.

[0084] Although nucleotide sequences which encode NHAP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring NHAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding NHAP possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding NHAP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

[0085] The invention also encompasses production of DNA sequences which encode NHAP and NHAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding NHAP or any fragment thereof.

[0086] Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, or a fragment of SEQ ID NO:4 under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0087] The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25° C., more preferably of at least about 42° C., and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

[0088] Methods for DNA sequencing and analysis are well known in the art. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway N.J.), or combinations of polymerases and proofreading exonucleases, such as those found in the ELONGASE amplification system (Invitrogen). Preferably, sequence preparation is automated with machines such as the HYDRA microdispenser (Robbins Scientific, Sunnyvale Calif.), MICROLAB 2200 (Hamilton, Reno Nev.), and the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). Machines used for sequencing include the ABI 3700, 377 or 373 DNA sequencing systems (ABI), the MEGABACE 1000 DNA sequencing system (APB), and the like. Sequences can be analyzed using computer programs and algorithms well known in the art. (See, e.g., Ausubel, supra, unit 7.7; and Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, Inc, New York, N.Y.)

[0089] The nucleic acid sequences encoding NHAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries to walk genomic DNA (Clontech). This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68° C. to 72° C.

[0090] When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5′ regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.

[0091] Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, ABI), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.

[0092] In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode NHAP may be cloned in recombinant DNA molecules that direct expression of NHAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express NHAP.

[0093] The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter NHAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

[0094] In another embodiment, sequences encoding NHAP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp. Ser. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids Symp. Ser. 7:225-232.) Alternatively, NHAP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (ABI). Additionally, the amino acid sequence of NHAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

[0095] The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, W H Freeman and Co., New York, N.Y.)

[0096] In order to express a biologically active NHAP, the nucleotide sequences encoding NHAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ untranslated regions in the vector and in polynucleotide sequences encoding NHAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding NHAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding NHAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0097] Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding NHAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and periodic supplements) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

[0098] A variety of expression vector/host systems may be utilized to contain and express sequences encoding NHAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed.

[0099] In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NHAP. For example, when cloning in bacterial systems, inducible promoters, e.g., hybrid lacZ promoter of the PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Invitrogen), may be used. Ligation of sequences encoding NHAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a calorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of NHAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of NHAP may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.

[0100] Yeast expression systems may be used for production of NHAP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) Methods Enzymol. 153:516-54; Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)

[0101] Plant systems may also be used for expression of NHAP. Transcription of sequences encoding NHAP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

[0102] In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding NHAP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses NHAP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.

[0103] Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.

[0104] For long term production of recombinant proteins in mammalian systems, stable expression of NHAP in cell lines is preferred. For example, sequences encoding NHAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

[0105] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., atithocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.), β glucuronidase and its substrate β-D-glucuronoside, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131.)

[0106] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding NHAP is inserted within a marker gene sequence, transformed cells containing sequences encoding NHAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding NHAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

[0107] In general, host cells that contain the nucleic acid sequence encoding NHAP and that express NHAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.

[0108] Immunological methods for detecting and measuring the expression of NHAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on NHAP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et al. (1997 and periodic supplements) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; and Maddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

[0109] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NHAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding NHAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0110] Host cells transformed with nucleotide sequences encoding NHAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode NHAP may be designed to contain signal sequences which direct secretion of NHAP through a prokaryotic or eukaryotic cell membrane.

[0111] In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.

[0112] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding NHAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric NHAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NHAP activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the NHAP encoding sequence and the heterologous protein sequence, so that NHAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.

[0113] In a further embodiment of the invention, synthesis of radiolabeled NHAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega, Madison, Wis.). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably ³⁵S-methionine.

[0114] Fragments of NHAP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431A peptide synthesizer (ABI). Various fragments of NHAP may be synthesized separately and then combined to produce the full length molecule.

THERAPEUTICS

[0115] Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between NHAP and an aspartic protease-like protein from mouse (GI 1906890). In addition, NHAP is expressed in endocrine tissues, cancer, inflammation and the immune response, and respiratory disorders. Therefore, NHAP appears to play a role in respiratory, endocrinological, and immunological disorders, and cancer.

[0116] Therefore, in one embodiment, NHAP or a fragment or derivative thereof may be administered to a subject to treat or prevent an endocrinological disorder associated with decreased expression or activity of NHAP. Such disorders can include, but are not limited to, disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; and disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism.

[0117] In another embodiment, a vector capable of expressing NHAP or a fragment or derivative thereof may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those described above.

[0118] In a further embodiment, a pharmaceutical composition comprising a substantially purified NHAP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those provided above.

[0119] In still another embodiment, an agonist which modulates the activity of NHAP may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those listed above.

[0120] In a further embodiment, an antagonist of NHAP may be administered to a subject to treat or prevent an endocrinological disorder associated with increased expression or activity of NHAP. Such disorders can include, but are not limited to, disorders associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH); disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; and disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia). In one aspect, an antibody which specifically binds NHAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express NHAP.

[0121] In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NHAP may be administered to a subject to treat or prevent an endocrinological disorder including, but not limited to, those described above.

[0122] In a further embodiment, an antagonist of NHAP may be administered to a subject to treat or prevent a respiratory disorder. Such disorders can include, but are not limited to, allergy, asthma, acute and chronic inflammatory lung diseases, Adult Respiratory Distress Syndrome (ARDS), emphysema, pulmonary congestion and edema, Chronic Obstructive Pulmonary Disease (COPD), interstitial lung diseases, and lung cancers.

[0123] In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NHAP may be administered to a subject to treat or prevent a respiratory disorder including, but not limited to, those described above.

[0124] In a further embodiment, an antagonist of NHAP may be administered to a subject to treat or prevent a cancer. Such a cancer may include, but is not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

[0125] In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NHAP may be administered to a subject to treat or prevent a cancer including, but not limited to, those described above.

[0126] In a further embodiment, an antagonist of NHAP may be administered to a subject to treat or prevent an immunological disorder. Such disorders may include, but are not limited to, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

[0127] In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NHAP may be administered to a subject to treat or prevent an immunological disorder including, but not limited to, those described above.

[0128] In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0129] An antagonist of NHAP may be produced using methods which are generally known in the art. In particular, purified NHAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind NHAP. Antibodies to NHAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.

[0130] For the production of polyclonal antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with NHAP or with any fragment or oligopeptide thereof which has immunogenic properties. Rats and mice are preferred hosts for downstream applications involving monoclonal antibody production. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable. (For review of methods for antibody production and analysis, see, e.g., Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)

[0131] It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to NHAP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 14 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of NHAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.

[0132] Monoclonal antibodies to NHAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0133] In addition, techniques developed for the production of “chimeric antibodies,” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce NHAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)

[0134] Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

[0135] Antibody fragments which contain specific binding sites for NHAP may also be generated. For example, such fragments include, but are not limited to, F(ab′)₂ fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science 246:1275-1281.)

[0136] Various immunoassays may be used for screening to identify antibodies having the desired specificity and minimal cross-reactivity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between NHAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NHAP epitopes is preferred, but a competitive binding assay may also be employed. (Maddox, supra.)

[0137] Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for NHAP. Affinity is expressed as an association constant, K_(a), which is defined as the molar concentration of NHAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K_(a) determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple NHAP epitopes, represents the average affinity, or avidity, of the antibodies for NHAP. The K_(a) determined for a preparation of monoclonal antibodies, which are monospecific for a particular NHAP epitope, represents a true measure of affinity. High-affinity antibody preparations with K_(a) ranging from about 10⁹ to 10¹² L/mole are preferred for use in immunoassays in which the NHAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to 10⁷ L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of NHAP, preferably in active form, from the antibody. (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington, D. C.; and Liddell, J. E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York, N.Y.)

[0138] The titre and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is preferred for use in procedures requiring precipitation of NHAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)

[0139] In another embodiment of the invention, the polynucleotides encoding NHAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding NHAP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding NHAP. Thus, complementary molecules or fragments may be used to modulate NHAP activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NHAP.

[0140] Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding NHAP. (See, e.g., Sambrook, supra; and Ausubel, supra.)

[0141] Genes encoding NHAP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding NHAP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.

[0142] As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the gene encoding NHAP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

[0143] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding NHAP.

[0144] Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0145] Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding NHAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.

[0146] RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

[0147] Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)

[0148] Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

[0149] An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of NHAP, antibodies to NHAP, and mimetics, agonists, antagonists, or inhibitors of NHAP. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.

[0150] The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0151] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

[0152] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[0153] Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.

[0154] Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

[0155] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

[0156] Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[0157] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0158] The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0159] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0160] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of NHAP, such labeling would include amount, frequency, and method of administration.

[0161] Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

[0162] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0163] A therapeutically effective dose refers to that amount of active ingredient, for example NHAP or fragments thereof, antibodies of NHAP, and agonists, antagonists or inhibitors of NHAP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₅₀ (the dose therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to thereapeituc effects is the therapeutic index, and it can be expressed as the LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

[0164] The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

[0165] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

DIAGNOSTICS

[0166] In another embodiment, antibodies which specifically bind NHAP may be used for the diagnosis of disorders characterized by expression of NHAP, or in assays to monitor patients being treated with NHAP or agonists, antagonists, or inhibitors of NHAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NHAP include methods which utilize the antibody and a label to detect NHAP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.

[0167] A variety of protocols for measuring NHAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NHAP expression. Normal or standard values for NHAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to NHAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of NHAP expressed in subject samples, control and disease, from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

[0168] In another embodiment of the invention, the polynucleotides encoding NHAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of NHAP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of NHAP, and to monitor regulation of NHAP levels during therapeutic intervention.

[0169] In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding NHAP or closely related molecules may be used to identify nucleic acid sequences which encode NHAP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding NHAP, allelic variants, or related sequences.

[0170] Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the NHAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:2, SEQ ID NO:4 or from genomic sequences including promoters, enhancers, and introns of the NHAP gene.

[0171] Means for producing specific hybridization probes for DNAs encoding NHAP include the cloning of polynucleotide sequences encoding NHAP or NHAP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

[0172] Polynucleotide sequences encoding NHAP may be used for the diagnosis of a disorder associated with expression of NHAP. Examples of such a disorder include, but are not limited to, endocrinological disorders such as disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH); and disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism; disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; and disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia); respiratory disorders such as allergy, asthma, acute and chronic inflammatory lung diseases, ARDS, emphysema, pulmonary congestion and edema, COPD, interstitial lung diseases, and lung cancers; cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and immunological disorders such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. The polynucleotide sequences encoding NHAP may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered NHAP expression. Such qualitative or quantitative methods are well known in the art.

[0173] In a particular aspect, the nucleotide sequences encoding NHAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding NHAP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding NHAP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

[0174] In order to provide a basis for the diagnosis of a disorder associated with expression of NHAP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding NHAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.

[0175] Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

[0176] With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

[0177] Additional diagnostic uses for oligonucleotides designed from the sequences encoding NHAP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding NHAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding NHAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.

[0178] Methods which may also be used to quantitate the expression of NHAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

[0179] In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.

[0180] Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0181] In another embodiment of the invention, nucleic acid sequences encoding NHAP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)

[0182] Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding NHAP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.

[0183] In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

[0184] In another embodiment of the invention, NHAP, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between NHAP and the agent being tested may be measured.

[0185] Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with NHAP, or fragments thereof, and washed. Bound NHAP is then detected by methods well known in the art. Purified NHAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

[0186] In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding NHAP specifically compete with a test compound for binding NHAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with NHAP.

[0187] In additional embodiments, the nucleotide sequences which encode NHAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

[0188] The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.

EXAMPLES

[0189] I. Construction of cDNA Libraries

[0190] RNA was purchased from Clontech (Palo Alto, Calif.) or isolated at Incyte from tissues described in Table 1. The tissue was homogenized and lysed in guanidinium isothiocyanate, and the lysate was centrifuged over a CsCl cushion. Alternatively, the tissue was homogenized and lysed in phenol or a suitable mixture of denaturants such as TRIZOL reagent (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate, and the lysate was extracted with chloroform (1:5 v/v). RNA was precipitated from lysates with either isopropanol or sodium acetate and ethanol. Alternatively, RNA was purified from lysates by preparative agarose gel electrophoresis and recovered from Whatman P81 paper (Whatman, Lexington, Mass.). Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity, and RNA was maintained in RNase-free solutions. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega, Madison, Wis.), Oligotex resin, or the OLIGOTEX kit (Qiagen, Valencia Calif.). Alternatively, RNA was isolated directly from tissue lysates using the RNA Isolation kit (Stratagene) or the Ambion PolyA Quick kit (Ambion, Austin, Tex.).

[0191] RNA was used for cDNA synthesis and construction of the cDNA libraries according to procedures recommended in the UNIZAP vector (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), both of which are based on methods well known in the art (Ausubel, 1997, units 5.1-6.6). Alternatively, cDNA libraries were constructed by Stratagene using RNA provided by Incyte. Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and cDNA was digested with an appropriate restriction enzyme(s). For most libraries, cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000 or SEPHAROSE CL-2B or CL4B column chromatography (APB) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., pBluescript (Stratagene), PSPORT1 (Invitrogen), pINCY (Incyte Genomics Inc, Palo Alto, Calif.). pINCY was amplified in JM109 cells and purified using the QIAQUICK column (QIAGEN Inc). Recombinant plasmids were transformed into competent E. coli cells, e.g., XL1-Blue, XL1-BlueMRF, or SOLR (Stratagene) or DH5α, DH10B, or ELECTROMAX DH10B cells (Invitrogen).

[0192] II. Isolation of cDNA Clones

[0193] Plasmids were recovered from host cells by in vivo excision (UNIZAP vector system, Stratagene) or by cell lysis. Plasmids were purified using the MINIPREP kit (Edge Biosystems, Gaithersburg Md.); QIAwell-8 Plasmid, QIAwell PLUS DNA, or QIAwell ULTRA DNA purification systems; or REAL Prep 96 plasmid kit (QIAGEN Inc) using the recommended protocol. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.

[0194] Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR (Rao, V. B. (1994) Anal. Biochem. 216:1-14) in a high-throughput format. Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates (Genetix Ltd, Christchurch UK) and concentration of amplified plasmid DNA was quantified fluorometrically using Pico Green Dye (Molecular Probes, Eugene Oreg.) and a Fluoroscan II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

[0195] III. Sequencing and Analysis

[0196] The cDNAs were prepared for sequencing using either an ABI CATALYST 800 (ABI) or a Hamilton MICRO LAB 2200 (Hamilton, Reno, Nev.) in combination with the DNA ENGINE thermal cyclers (MJ Research). The cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM 377 sequencing system (ABI). Alternatively, cDNAs were prepared and sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frame was determined using standard methods (Ausubel, supra).

[0197] The nucleotide sequences and/or amino acid sequences of the Sequence Listing were queried against databases such as GenBank primate (pri), rodent (rod), mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS, and other databases which contain previously identified and annotated motifs and sequences. Algorithms such as Smith Waterman which deal with primary sequence patterns and secondary structure gap penalties (Smith, T. et al. (1992) Protein Engineering 5:35-51) and programs and algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol. 215:403-410), and HMM (Hidden Markov Models; Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365 and Sonnhammer, E. L. L. et al. (1997) Proteins 28:405-420) were used to assemble and analyze nucleotide and amino acid sequences. The databases, programs, algorithms, methods and tools are available, well known in the art, and described in Ausubel (supra, unit 7.7), in Meyers, R. A. (1995; Molecular Biology and Biotechnoloy, Wiley VCH, Inc, New York N.Y., p 856-853), in documentation provided with software (Genetics Computer Group (GCG), Madison Wis.), and on the world wide web (www). Two comprehensive websites which list, describe, and/or link many of the databases and tools are: 1) the www resource in practical sequence analysis (http://genome.wustl.edu/), and 2) the bibliography of computational gene recognition (http://linkage.rockefeller.edu/wli/gene/ programs.html). For example, the first website links PFAM as a database (http://genome.wustl.edu/Pfam/) and as an HMM search tool (http://genome.wustl.edu/eddy/cgi-bin/hmm_page.cgi). Table 2 summarizes the databases and tools used herein. The first column of Table 2 shows the tool, program, or algorithm; the second column, the database; the third column, a brief description; and the fourth column (where applicable), scores for determining the strength of a match between two sequences (the higher the value, the more homologous).

[0198] IV. Cloning of Full Length NHAP

[0199] The GENETRAPPER cDNA Positive Selection System kit (Invitrogen) was employed to isolate full length cDNA clones of NHAP-1 and NHAP-2. Following the manufacturer's instructions, oligonucleotides were designed based on partial nucleic acid sequences from Incyte clone 2756549, biotinylated at the 3′ end, and hybridized to single stranded DNA from plasmid cDNA libraries of human lung (Cat. No. 10424-018, Invitrogen) and human leukocytes (Cat. No. 10421-014, Invitrogen). Five cDNA clones; gt83, gt86, gt97, gt88, and gt91 were isolated from lung cDNA library, and five cDNA clones; gt4, gt22, gt49, gt53, and gt90 were isolated from the leukocyte library. Sequencing revealed that the clones isolated from the lung library were identical in nucleic acid sequence to Incyte clones 372637 and 1242901 and to the gene subsequently named NHAP-1 (HUPM-4 in the prior application). However, the clones isolated from the lung library differed in nucleic acid sequences from those isolated from the leukocyte library and from Incyte clones 2435410 and 2756549. Thus two genes were identified and were subsequently named NHAP-1 and NHAP-2. NHAP-1 encompasses cDNA clones gt83, gt86, gt97, gt88, gt91, Incyte clone 372637 and 1242901. NHAP-2 encompasses cDNA clones gt4, gt22, gt49, gt53, gt90 and Incyte clones 2435410 and 2756549. Sequence homology analysis showed 89% nucleic acid identity between NHAP-1 and NHAP-2.

[0200] V. Northern Analysis

[0201] Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)

[0202] Membrane-based northern analysis was perfonned on RNA samples from a variety of human tissues using Multiple Tissue Northern Blots (Clontech) probed with NHAP-2 cDNA. The probe was labeled with ³³P using the random primer labeling method with the HIGH PRIMER DNA labeling kit (Boerheinger Mannheim, Indianapolis, Ind.). Hybridization was conducted under high stringency conditions in a solution containing 50% formamide, 5× SSC, 50 mM NaPO4, pH 7.4, 1× Denhardts, 2% SDS and 100 ug/ml Salmon Sperm DNA at 42° C. overnight. The blots were washed with 2× SSC at room temperature 2-3 times, followed, if necessary, by washes with 0.2× SSC, 0.1% SDS at 50° C. 1-2 times, and subjected to autoradiogrphy at −80° C. The northern analysis demonstrated a high level of expression of an RNA species of ˜1.3 kb from kidney, peripheral blood leukocytes, spleen and lymph nodes (FIG. 4). This RNA species was also expressed at a lower level in lung, bone marrow, thymus, and fetal liver. Since NHAP-2 has 89% homology to NHAP-1, the northern analysis reflected the expression profile of both NHAP-1 and NHAP-2. When the above blots were stripped and reprobed with NHAP-1-specific oligonucleotide, the expression of the 1.3 kb RNA species was found only in the lung (FIG. 5).

[0203] Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ database (Incyte Genomics).

[0204] The basis of the search is the product score, which is defined as: $\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0205] The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.

[0206] The results of Northern analysis showed the transcript encoding NHAP in a variety of cDNA libraries, at least 59% of which involve cancer and immortalized cell lines, and at least 22% of which involve inflammation and the immune response. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library.

[0207] VI. Labeling and Use of Individual Hybridization Probes

[0208] Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:4 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (APB). An aliquot containing 10⁷ counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN).

[0209] The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1× saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.

[0210] VII. Microarrays

[0211] A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, WV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.

[0212] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; and Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.

[0213] VIII. Complementary Polynucleotides

[0214] Sequences complementary to the NHAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring NHAP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software and the coding sequence of NHAP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the NHAP-encoding transcript.

[0215] IX. Expression of NHAP-1

[0216] The cDNA encoding NHAP-1 was used to express full-length NHAP-1 by subcloning the cDNAs into appropriate vectors and introducing the constructs into host cells. For expression of NHAP-1 in E. coli, NHAP-1 cDNA was subcloned into a bacterial expression vector pET15b (Novagen, Inc., Madison, Wis.) which provides an N-terminal Tag of His6. To monitor expression in E. coli, the cell lysates from cultures before and after IPTG induction were separated using polyacrylamide gel electrophoresis under reduced denatured conditions, and probed with preimmune and immune serums (IC620). Binding of the antisera was detected by HRP-conjugated donkey anti-rabbit Ig and visualized using ECL (enhanced chemiluminescence) system (Amersham Pharmacia Biotech). NHAP-1 recombinant protein was detected as a ˜45 kd band predominantly from the insoluble fraction in IPTG-induced cells exposed to immune serum (FIG. 6). A band was not detected in uninduced cells or cells probed with preimmune serum. NHAP-1 cDNA was also subcloned into the baculovirus pFast-bac-HTc (Invitrogen) for expression in Sf9 insect cells, and into pCMV-SPORT (Invitrogen) for expression in mammalian HEK 293 cells.

[0217] X. Demonstration of NHAP Activity

[0218] Protease activity of NHAP is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantitated by spectrophotometric (or fluorometric) absorption of the released chromophore. (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York, N.Y., pp.25-55) Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases), animopeptidase (leucine aminopeptidase), or carboxypeptidase (carboxypeptidase A and B, procollagen C-proteinase). Chromogens commonly used are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid. Assays are performed at ambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette and followed by the increase/decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate. The change in absorbance is proportional to the enzyme activity in the assay.

[0219] XI. Functional Assays

[0220] NHAP function is assessed by expressing the sequences encoding NHAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include pCMV SPORT and pCR™ 3.1 (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech,), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York, N.Y.

[0221] The influence of NHAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding NHAP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding NHAP and other genes of interest can be analyzed by Northern analysis or microarray techniques.

[0222] XII. Production of NHAP Specific Antibodies

[0223] An oligopeptide containing 19 amino acid residues from the C-terminus of NHAP-1 was synthesized. Two rabbits were immunized with the oligopeptide-KLH complex in complete Freund's adjuvant (Zeneca LifeScience Molecules, Wilmington, Del.). The resulting antisera, IC619 and IC620, were tested for antipeptide activity by ELISA. Both antisera recognized recombinant protein expressed in E. coli and in Sf9 insect cells by western blot analysis. Briefly, E. coli and Sf9 cells containing the corresponding expression constructs were lysed, and proteins were separated on a denatured PAGE gel (NuPage gels, Novex) and transferred onto a nitrocellulose membrane according to the method previously described. The blot was then probed with antisera IC619 or IC620. Binding of the antisera was detected by HRP-conjugated donkey anti-rabbit Ig and visualized using ECL (enhanced chemiluminescence) system (Amersham Pharmacia Biotech).

[0224] XIII. Purification of Naturally Occurring NHAP Using Specific Antibodies

[0225] Naturally occurring or recombinant NHAP is substantially purified by immunoaffinity chromatography using antibodies specific for NHAP. An immunoaffinity column is constructed by covalently coupling anti-NHAP antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

[0226] Media containing NHAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of NHAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/NHAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and NHAP is collected.

[0227] XIV Immunocytochemical Analysis of NHAP-1 in Normal and Disease Tissues

[0228] Immunocytochemical analysis was performed to determine protein localization in human tissue samples using NHAP-1-specific rabbit immune serum IC619 as the primary antibody. The analysis was performed by LifeSpan BioSciences, Inc., Seattle Wash. The detection system consisted of a DAKO LSAB+Kit (DAKO corp., Carpinteria Calif.) containing labelled Streptavidin-Biotin Kit with a biotinylated secondary antibody followed by application of a streptavidin-horseradish peroxidase conjugate and DAB substrate. Tissues were also blocked for endogenous biotin and endogenous peroxide. Negative controls performed on each tissue sample included staining with pre-immune sera. In addition, experiments were performed to block staining by incubating Antibody IC619 with a 10 fold excess of immunizing peptide derivedfrom NAP1. The analysis demonstrated that antibody IC619 produced strong positive staining in the anterior lobe of the pituitary, in thyroid follicular cells and within the Type II pneumocytes of the lung. In all lung tissues examined, both normal and diseased, Type II pneumocytes stained positive for antibody IC619. In particular, the bronchioloalveolar carcinoma and lung adenocarcinoma produced strong positive staining. Other lung neoplasms including a small cell, epidermoid cell, adenocarcinoma and metastatic colon adenocarcinoma were negative when stained.

[0229] XV. Chromosome Localization of NHAP-1 and NHAP-2 by Fluorescence In Situ Hybridization (FISH) Analysis

[0230] FISH analysis was performed to determine chromosomal localization of both NHAP-1 and NHAP-2 (Genome Systems, Inc., St. Louis, Mo.). DNA from two genomic clones, corresponding to NHAP-1 and NHAP-2, were labeled with digoxigenin dUTP by nick translation. Labeled probes were combined with sheared human DNA and independently hybridized to normal metaphase chromosomes derived from PHA stimulated peripheral blood lymphocytes from a male donor in a solution containing 50% formamide, 10% dextran sulfate and 2× SSC. Specific signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with DAPI. These experiments resulted in the specific labeling of the long arms of chromosome 19. Quantification of 10 spreads with specific hybridization to chromosome 19 demonstrated that the genes encoding NHAP-1 and NHAP-2 are indistinguishable from each other and are located at a position which is 73% of the distance from the centromere to the telomere of chromosome arm 19q, an area that corresponds to band 19q13.3.

[0231] XVI. Identification of Molecules Which Interact with NHAP

[0232] NHAP, or biologically active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled NHAP, washed, and any wells with labeled NHAP complex are assayed. Data obtained using different concentrations of NHAP are used to calculate values for the number, affinity, and association of NHAP with the candidate molecules.

[0233] Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

1 24 63 amino acids amino acid single linear BMARNOT02 135360 1 Met Asp Ile Leu Ile Cys Thr Asp Phe Gly Ser Val Asn Tyr Phe 5 10 15 Asn Val Trp Arg Leu Pro Lys Ser Tyr Leu Ser Leu Phe Tyr Ser 20 25 30 Arg Ile Tyr Ile Val His Asp Glu Val Lys Asp Lys Ala Phe Glu 35 40 45 Leu Glu Leu Ser Trp Val Gly Glu Cys Lys Leu Phe Leu Tyr Ile 50 55 60 Tyr Leu Pro 262 amino acids amino acid single linear TLYMNOT02 447484 2 Met Gly Arg Glu Ile Arg Ser Glu Glu Pro Glu Glu Ser Val Pro 5 10 15 Phe Ser Cys Asp Trp Arg Lys Val Ala Gly Ala Ile Ser Pro Ile 20 25 30 Lys Asp Gln Lys Asn Cys Asn Cys Cys Trp Ala Met Ala Ala Ala 35 40 45 Gly Asn Ile Glu Thr Leu Trp Arg Ile Ser Phe Trp Asp Phe Val 50 55 60 Asp Val Ser Val Gln Glu Leu Leu Asp Cys Gly Arg Cys Gly Asp 65 70 75 Gly Cys His Gly Gly Phe Val Trp Asp Ala Phe Ile Thr Val Leu 80 85 90 Asn Asn Ser Gly Leu Ala Ser Glu Lys Asp Tyr Pro Phe Gln Gly 95 100 105 Lys Val Arg Ala His Arg Cys His Pro Lys Lys Tyr Gln Lys Val 110 115 120 Ala Trp Ile Gln Asp Phe Ile Met Leu Gln Asn Asn Glu His Arg 125 130 135 Ile Ala Gln Tyr Leu Ala Thr Tyr Gly Pro Ile Thr Val Thr Ile 140 145 150 Asn Met Lys Pro Leu Gln Leu Tyr Arg Lys Gly Val Ile Lys Ala 155 160 165 Thr Pro Thr Thr Cys Asp Pro Gln Leu Val Asp His Ser Val Leu 170 175 180 Leu Val Gly Phe Gly Ser Val Lys Ser Glu Glu Gly Ile Trp Ala 185 190 195 Glu Thr Val Ser Ser Gln Ser Gln Pro Gln Pro Pro His Pro Thr 200 205 210 Pro Tyr Trp Ile Leu Lys Asn Ser Trp Gly Ala Gln Trp Gly Glu 215 220 225 Lys Gly Tyr Phe Arg Leu His Arg Gly Ser Asn Thr Cys Gly Ile 230 235 240 Thr Lys Phe Pro Leu Thr Ala Arg Val Gln Lys Pro Asp Met Lys 245 250 255 Pro Arg Val Ser Cys Pro Pro 260 314 amino acids amino acid single linear PROSTUT03 789927 3 Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg 5 10 15 Ala Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu Ser 20 25 30 Gly Pro Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly 35 40 45 Glu Asp Ala Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg 50 55 60 Leu Trp Asp Ser His Val Cys Gly Val Ser Leu Leu Ser His Arg 65 70 75 Trp Ala Leu Thr Ala Ala His Cys Phe Glu Thr Tyr Ser Asp Leu 80 85 90 Ser Asp Pro Ser Gly Trp Met Val Gln Phe Gly Gln Leu Thr Ser 95 100 105 Met Pro Ser Phe Trp Ser Leu Gln Ala Tyr Tyr Thr Arg Tyr Phe 110 115 120 Val Ser Asn Ile Tyr Leu Ser Pro Arg Tyr Leu Gly Asn Ser Pro 125 130 135 Tyr Asp Ile Ala Leu Val Lys Leu Ser Ala Pro Val Thr Tyr Thr 140 145 150 Lys His Ile Gln Pro Ile Cys Leu Gln Ala Ser Thr Phe Glu Phe 155 160 165 Glu Asn Arg Thr Asp Cys Trp Val Thr Gly Trp Gly Tyr Ile Lys 170 175 180 Glu Asp Glu Ala Leu Pro Ser Pro His Thr Leu Gln Glu Val Gln 185 190 195 Val Ala Ile Ile Asn Asn Ser Met Cys Asn His Leu Phe Leu Lys 200 205 210 Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp Met Val Cys Ala Gly 215 220 225 Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly Asp Ser Gly Gly 230 235 240 Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln Ile Gly Val 245 250 255 Val Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg Pro Gly Val 260 265 270 Tyr Thr Asn Ile Ser His His Phe Glu Trp Ile Gln Lys Leu Met 275 280 285 Ala Gln Ser Gly Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu 290 295 300 Phe Phe Pro Leu Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 305 310 420 amino acids amino acid single linear LUNGAST01 877617 4 Met Ser Pro Pro Pro Leu Leu Gln Pro Leu Leu Leu Leu Leu Pro 5 10 15 Leu Leu Asn Val Glu Pro Ser Gly Ala Thr Leu Ile Arg Ile Pro 20 25 30 Leu His Arg Val Gln Pro Gly Arg Arg Thr Leu Asn Leu Leu Arg 35 40 45 Gly Trp Arg Glu Pro Ala Glu Leu Pro Lys Leu Gly Ala Pro Ser 50 55 60 Pro Gly Asp Lys Pro Ile Phe Val Pro Leu Ser Asn Tyr Arg Asp 65 70 75 Val Gln Tyr Phe Gly Glu Ile Gly Leu Gly Thr Pro Pro Gln Asn 80 85 90 Phe Thr Val Ala Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro 95 100 105 Ser Arg Arg Cys His Phe Phe Ser Val Pro Cys Trp Leu His His 110 115 120 Arg Phe Asp Pro Lys Ala Ser Ser Ser Phe Gln Ala Asn Gly Thr 125 130 135 Lys Phe Ala Ile Gln Tyr Gly Thr Gly Arg Val Asp Gly Ile Leu 140 145 150 Ser Glu Asp Lys Leu Thr Ile Gly Gly Ile Lys Gly Ala Ser Val 155 160 165 Ile Phe Gly Glu Ala Leu Trp Glu Pro Ser Leu Val Phe Ala Phe 170 175 180 Ala His Phe Asp Gly Ile Leu Gly Leu Gly Phe Pro Ile Leu Ser 185 190 195 Val Glu Gly Val Arg Pro Pro Met Asp Val Leu Val Glu Gln Gly 200 205 210 Leu Leu Asp Lys Pro Val Phe Ser Phe Tyr Leu Asn Arg Asp Pro 215 220 225 Glu Glu Pro Asp Gly Gly Glu Leu Val Leu Gly Gly Ser Asp Pro 230 235 240 Ala His Tyr Ile Pro Pro Leu Thr Phe Val Pro Val Thr Val Pro 245 250 255 Ala Tyr Trp Gln Ile His Met Glu Arg Val Lys Val Gly Pro Gly 260 265 270 Leu Thr Leu Cys Ala Lys Gly Cys Ala Ala Ile Leu Asp Thr Gly 275 280 285 Thr Ser Leu Ile Thr Gly Pro Thr Glu Glu Ile Arg Ala Leu His 290 295 300 Ala Ala Ile Gly Gly Ile Pro Leu Leu Ala Gly Glu Tyr Ile Ile 305 310 315 Leu Cys Ser Glu Ile Pro Lys Leu Pro Ala Val Ser Phe Leu Leu 320 325 330 Gly Gly Val Trp Phe Asn Leu Thr Ala His Asp Tyr Val Ile Gln 335 340 345 Thr Thr Arg Asn Gly Val Arg Leu Cys Leu Ser Gly Phe Gln Ala 350 355 360 Leu Asp Val Pro Pro Pro Ala Gly Pro Phe Trp Ile Leu Gly Asp 365 370 375 Val Phe Leu Gly Thr Tyr Val Ala Val Phe Asp Arg Gly Asp Met 380 385 390 Lys Ser Ser Ala Arg Val Gly Leu Ala Arg Ala Arg Thr Arg Gly 395 400 405 Ala Asp Leu Gly Trp Gly Glu Thr Ala Gln Ala Gln Phe Pro Gly 410 415 420 200 amino acids amino acid single linear KIDNTUT01 999322 5 Met Cys Glu Leu Met Tyr His Leu Gly Glu Pro Ser Leu Ala Gly 5 10 15 Gln Arg Leu Ile Gln Asp Asp Met Leu Cys Ala Gly Ser Val Gln 20 25 30 Gly Lys Lys Asp Ser Cys Gln Val Thr Ala Ala Pro Gly His Pro 35 40 45 Ile Gln Leu Cys Gly Pro Phe Arg Leu Thr Leu Ser Trp Thr Phe 50 55 60 Ser Pro Cys Pro Thr Pro Gln Gly Leu Gln Arg Asp Gln Ser Pro 65 70 75 Cys Leu Ala Pro Trp Pro Gln Gln Leu Ile Leu Glu Gly Thr Trp 80 85 90 Gly Pro Gly Val Ser Leu Asn Ala Asp Leu Met Gly Pro Ser Leu 95 100 105 Ser Leu Pro Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Pro Ile 110 115 120 Asn Asp Thr Trp Ile Gln Ala Gly Ile Val Ser Trp Gly Phe Gly 125 130 135 Cys Ala Arg Pro Phe Arg Pro Gly Val Tyr Thr Gln Val Leu Ser 140 145 150 Tyr Thr Asp Trp Ile Gln Arg Thr Leu Ala Glu Ser His Ser Gly 155 160 165 Met Ser Gly Ala Arg Pro Gly Ala Pro Gly Ser His Ser Gly Thr 170 175 180 Ser Arg Ser His Pro Val Leu Leu Leu Glu Leu Leu Thr Val Cys 185 190 195 Leu Leu Gly Ser Leu 200 435 amino acids amino acid single linear COLNNOT13 1337018 6 Met Asp Pro Asp Ser Asp Gln Pro Leu Asn Ser Leu Asp Val Lys 5 10 15 Pro Leu Arg Lys Pro Arg Ile Pro Met Glu Thr Phe Arg Lys Val 20 25 30 Gly Ile Pro Ile Ile Ile Ala Leu Leu Ser Leu Ala Ser Ile Ile 35 40 45 Ile Val Val Val Leu Ile Lys Val Ile Leu Asp Lys Tyr Tyr Phe 50 55 60 Leu Cys Gly Gln Pro Leu His Phe Ile Pro Arg Lys Gln Leu Cys 65 70 75 Asp Gly Glu Leu Asp Cys Pro Leu Gly Glu Asp Glu Glu His Cys 80 85 90 Val Lys Ser Phe Pro Glu Gly Pro Ala Val Ala Val Arg Leu Ser 95 100 105 Lys Asp Arg Ser Thr Leu Gln Val Leu Asp Ser Ala Thr Gly Asn 110 115 120 Trp Phe Ser Ala Cys Phe Asp Asn Phe Thr Glu Ala Leu Ala Glu 125 130 135 Thr Ala Cys Arg Gln Met Gly Tyr Ser Ser Lys Pro Thr Phe Arg 140 145 150 Ala Val Glu Ile Gly Pro Asp Gln Asp Leu Asp Val Val Glu Ile 155 160 165 Thr Glu Asn Ser Gln Glu Leu Arg Met Arg Asn Ser Ser Gly Pro 170 175 180 Cys Leu Ser Gly Ser Leu Val Ser Leu His Cys Leu Ala Cys Gly 185 190 195 Glu Ser Leu Lys Thr Pro Arg Val Val Gly Gly Glu Glu Ala Ser 200 205 210 Val Asp Ser Trp Pro Trp Gln Val Ser Ile Gln Tyr Asp Lys Gln 215 220 225 His Val Cys Gly Gly Ser Ile Leu Asp Pro His Trp Val Leu Thr 230 235 240 Ala Ala His Cys Phe Arg Lys His Thr Asp Val Phe Asn Trp Lys 245 250 255 Val Arg Ala Gly Ser Asp Lys Leu Gly Ser Phe Pro Ser Leu Ala 260 265 270 Val Ala Lys Ile Ile Ile Ile Glu Phe Asn Pro Met Tyr Pro Lys 275 280 285 Asp Asn Asp Ile Ala Leu Met Lys Leu Gln Phe Pro Leu Thr Phe 290 295 300 Ser Gly Thr Val Arg Pro Ile Cys Leu Pro Phe Phe Asp Glu Glu 305 310 315 Leu Thr Pro Ala Thr Pro Leu Trp Ile Ile Gly Trp Gly Phe Thr 320 325 330 Lys Gln Asn Gly Gly Lys Met Ser Asp Ile Leu Leu Gln Ala Ser 335 340 345 Val Gln Val Ile Asp Ser Thr Arg Cys Asn Ala Asp Asp Ala Tyr 350 355 360 Gln Gly Glu Val Thr Glu Lys Met Met Cys Ala Gly Ile Pro Glu 365 370 375 Gly Gly Val Asp Thr Cys Gln Gly Asp Ser Gly Gly Pro Leu Met 380 385 390 Tyr Gln Ser Asp Gln Trp His Val Val Gly Ile Val Ser Trp Gly 395 400 405 Tyr Gly Cys Gly Gly Pro Ser Thr Pro Gly Val Tyr Thr Lys Val 410 415 420 Ser Ala Tyr Leu Asn Trp Ile Tyr Asn Val Trp Lys Ala Glu Leu 425 430 435 260 amino acids amino acid single linear COLNNOT27 1798496 7 Met Gly Arg Pro Arg Pro Arg Ala Ala Lys Thr Trp Met Phe Leu 5 10 15 Leu Leu Leu Gly Gly Ala Trp Ala Gly His Ser Arg Ala Gln Glu 20 25 30 Asp Lys Val Leu Gly Gly His Glu Cys Gln Pro His Ser Gln Pro 35 40 45 Trp Gln Ala Ala Leu Ser Gln Gly Gln Gln Leu Leu Cys Gly Gly 50 55 60 Val Leu Val Gly Gly Asn Trp Val Leu Thr Ala Ala His Cys Lys 65 70 75 Lys Pro Lys Tyr Thr Val Arg Leu Gly Asp His Ser Leu Gln Asn 80 85 90 Lys Asp Gly Pro Glu Gln Glu Ile Pro Val Val Gln Ser Ile Pro 95 100 105 His Pro Cys Tyr Asn Ser Ser Asp Val Glu Asp His Asn His Asp 110 115 120 Leu Met Leu Leu Gln Leu Arg Asp Gln Ala Ser Leu Gly Ser Lys 125 130 135 Val Lys Pro Ile Ser Leu Ala Asp His Cys Thr Gln Pro Gly Gln 140 145 150 Lys Cys Thr Val Ser Gly Trp Gly Thr Val Thr Ser Pro Arg Glu 155 160 165 Asn Phe Pro Asp Thr Leu Asn Cys Ala Glu Val Lys Ile Phe Pro 170 175 180 Gln Lys Lys Cys Glu Asp Ala Tyr Pro Gly Gln Ile Thr Asp Gly 185 190 195 Met Val Cys Ala Gly Ser Ser Lys Gly Ala Asp Thr Cys Gln Gly 200 205 210 Asp Ser Gly Gly Pro Leu Val Cys Asp Gly Ala Leu Gln Gly Ile 215 220 225 Thr Ser Trp Gly Ser Asp Pro Cys Gly Arg Ser Asp Lys Pro Gly 230 235 240 Val Tyr Thr Asn Ile Cys Arg Tyr Leu Asp Trp Ile Lys Lys Ile 245 250 255 Ile Gly Ser Lys Gly 260 175 amino acids amino acid single linear UTRSNOT08 2082147 8 Met Ala Gln Ser Gln Gly Trp Val Lys Arg Tyr Ile Lys Ala Phe 5 10 15 Cys Lys Gly Phe Phe Val Ala Val Pro Val Ala Val Thr Phe Leu 20 25 30 Asp Arg Val Ala Cys Val Ala Arg Val Glu Gly Ala Ser Met Gln 35 40 45 Pro Ser Leu Asn Pro Gly Gly Ser Gln Ser Ser Asp Val Val Leu 50 55 60 Leu Asn His Trp Lys Val Arg Asn Phe Glu Val His Arg Gly Asp 65 70 75 Ile Val Ser Leu Val Ser Pro Lys Asn Pro Glu Gln Lys Ile Ile 80 85 90 Lys Arg Val Ile Ala Leu Glu Gly Asp Ile Val Arg Thr Ile Gly 95 100 105 His Lys Asn Arg Tyr Val Lys Val Pro Arg Gly His Ile Trp Val 110 115 120 Glu Gly Asp His His Gly His Ser Phe Asp Ser Asn Ser Phe Gly 125 130 135 Pro Val Ser Leu Gly Leu Leu His Ala His Ala Thr His Ile Leu 140 145 150 Trp Pro Pro Glu Arg Trp Gln Lys Leu Glu Ser Val Leu Pro Pro 155 160 165 Glu Arg Leu Pro Val Gln Arg Glu Glu Glu 170 175 519 amino acids amino acid single linear ENDCNOT03 2170967 9 Met Phe Leu Leu Pro Leu Pro Ala Ala Gly Arg Val Val Val Arg 5 10 15 Arg Leu Ala Val Arg Arg Phe Gly Ser Arg Ser Leu Ser Thr Ala 20 25 30 Asp Met Thr Lys Gly Leu Val Leu Gly Ile Tyr Ser Lys Glu Lys 35 40 45 Glu Asp Asp Val Pro Gln Phe Thr Ser Ala Gly Glu Asn Phe Asp 50 55 60 Lys Leu Leu Ala Gly Lys Leu Arg Glu Thr Leu Asn Ile Ser Gly 65 70 75 Pro Pro Leu Lys Ala Gly Lys Thr Arg Thr Phe Tyr Gly Leu His 80 85 90 Gln Asp Phe Pro Ser Val Val Leu Val Gly Leu Gly Lys Lys Ala 95 100 105 Ala Gly Ile Asp Glu Gln Glu Asn Trp His Glu Gly Lys Glu Asn 110 115 120 Ile Arg Ala Ala Val Ala Ala Gly Cys Arg Gln Ile Gln Asp Leu 125 130 135 Glu Leu Ser Ser Val Glu Val Asp Pro Cys Gly Asp Ala Gln Ala 140 145 150 Ala Ala Glu Gly Ala Val Leu Gly Leu Tyr Glu Tyr Asp Asp Leu 155 160 165 Lys Gln Lys Lys Lys Met Ala Val Ser Ala Lys Leu Tyr Gly Ser 170 175 180 Gly Asp Gln Glu Ala Trp Gln Lys Gly Val Leu Phe Ala Ser Gly 185 190 195 Gln Asn Leu Ala Arg Gln Leu Met Glu Thr Pro Ala Asn Glu Met 200 205 210 Thr Pro Thr Arg Phe Ala Glu Ile Ile Glu Lys Asn Leu Lys Ser 215 220 225 Ala Ser Ser Lys Thr Glu Val His Ile Arg Pro Lys Ser Trp Ile 230 235 240 Glu Glu Gln Ala Met Gly Ser Phe Leu Ser Val Ala Lys Gly Ser 245 250 255 Asp Glu Pro Pro Val Phe Leu Glu Ile His Tyr Lys Gly Ser Pro 260 265 270 Asn Ala Asn Glu Pro Pro Leu Val Phe Val Gly Lys Gly Ile Thr 275 280 285 Phe Asp Ser Gly Gly Ile Ser Ile Lys Ala Ser Ala Asn Met Asp 290 295 300 Leu Met Arg Ala Asp Met Gly Gly Ala Ala Thr Ile Cys Ser Ala 305 310 315 Ile Val Ser Ala Ala Lys Leu Asn Leu Pro Ile Asn Ile Ile Gly 320 325 330 Leu Ala Pro Leu Cys Glu Asn Met Pro Ser Gly Lys Ala Asn Lys 335 340 345 Pro Gly Asp Val Val Arg Ala Lys Asn Gly Lys Thr Ile Gln Val 350 355 360 Asp Asn Thr Asp Ala Glu Gly Arg Leu Ile Leu Ala Asp Ala Leu 365 370 375 Cys Tyr Ala His Thr Phe Asn Pro Lys Val Ile Leu Asn Ala Ala 380 385 390 Thr Leu Thr Gly Ala Met Asp Val Ala Leu Gly Ser Gly Ala Thr 395 400 405 Gly Val Phe Thr Asn Ser Ser Trp Leu Trp Asn Lys Leu Phe Glu 410 415 420 Ala Ser Ile Glu Thr Gly Asp Arg Val Trp Arg Met Pro Leu Phe 425 430 435 Glu His Tyr Thr Arg Gln Val Val Asp Cys Gln Leu Ala Asp Val 440 445 450 Asn Asn Ile Gly Lys Tyr Arg Ser Ala Gly Ala Cys Thr Ala Ala 455 460 465 Ala Phe Leu Lys Glu Phe Val Thr His Pro Lys Trp Ala His Leu 470 475 480 Asp Ile Ala Gly Val Met Thr Asn Lys Asp Glu Val Pro Tyr Leu 485 490 495 Arg Lys Gly Met Thr Gly Arg Pro Thr Arg Thr Leu Ile Glu Phe 500 505 510 Leu Leu Arg Phe Ser Gln Asp Asn Ala 515 327 amino acids amino acid single linear SMCANOT01 2484218 10 Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Thr Asn Gly Thr Gly 5 10 15 Gly Ser Ser Gly Met Glu Val Asp Ala Ala Val Val Pro Ser Val 20 25 30 Met Ala Cys Gly Val Thr Gly Ser Val Ser Val Ala Leu His Pro 35 40 45 Leu Val Ile Leu Asn Ile Ser Asp His Trp Ile Arg Met Arg Ser 50 55 60 Gln Glu Gly Arg Pro Val Gln Val Ile Gly Ala Leu Ile Gly Lys 65 70 75 Gln Glu Gly Arg Asn Ile Glu Val Met Asn Ser Phe Glu Leu Leu 80 85 90 Ser His Thr Val Glu Glu Lys Ile Ile Ile Asp Lys Glu Tyr Tyr 95 100 105 Tyr Thr Lys Glu Glu Gln Phe Lys Gln Val Phe Lys Glu Leu Glu 110 115 120 Phe Leu Gly Trp Tyr Thr Thr Gly Gly Pro Pro Asp Pro Ser Asp 125 130 135 Ile His Val His Lys Gln Val Cys Glu Ile Ile Glu Ser Pro Leu 140 145 150 Phe Leu Lys Leu Asn Pro Met Thr Lys His Thr Asp Leu Pro Val 155 160 165 Ser Val Phe Glu Ser Val Ile Asp Ile Ile Asn Gly Glu Ala Thr 170 175 180 Met Leu Phe Ala Glu Leu Thr Tyr Thr Leu Ala Thr Glu Glu Ala 185 190 195 Glu Arg Ile Gly Val Asp His Val Ala Arg Met Thr Ala Thr Gly 200 205 210 Ser Gly Glu Asn Ser Thr Val Ala Glu His Leu Ile Ala Gln His 215 220 225 Ser Ala Ile Lys Met Leu His Ser Arg Val Lys Leu Ile Leu Glu 230 235 240 Tyr Val Lys Ala Ser Glu Ala Gly Glu Val Pro Phe Asn His Glu 245 250 255 Ile Leu Arg Glu Ala Tyr Ala Leu Cys His Cys Leu Pro Val Leu 260 265 270 Ser Thr Asp Lys Phe Lys Thr Asp Phe Tyr Asp Gln Cys Asn Asp 275 280 285 Val Gly Leu Met Ala Tyr Leu Gly Thr Ile Thr Lys Thr Cys Asn 290 295 300 Thr Met Asn Gln Phe Val Asn Lys Phe Asn Val Leu Tyr Asp Arg 305 310 315 Gln Gly Ile Gly Arg Arg Met Arg Gly Leu Phe Phe 320 325 458 amino acids amino acid single linear SINIUCT01 2680548 11 Met Ala Ala Pro Arg Ala Gly Arg Gly Ala Gly Trp Ser Leu Arg 5 10 15 Ala Trp Arg Ala Leu Gly Gly Ile Arg Trp Gly Arg Arg Pro Arg 20 25 30 Leu Thr Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Ser Asp 35 40 45 Pro Arg Ala Arg Val Thr Tyr Gly Thr Pro Ser Leu Trp Ala Arg 50 55 60 Leu Ser Val Gly Val Thr Glu Pro Arg Ala Cys Leu Thr Ser Gly 65 70 75 Thr Pro Gly Pro Arg Ala Gln Leu Thr Ala Val Thr Pro Asp Thr 80 85 90 Arg Thr Arg Glu Ala Ser Glu Asn Ser Gly Thr Arg Ser Arg Ala 95 100 105 Trp Leu Ala Val Ala Leu Gly Ala Gly Gly Ala Val Leu Leu Leu 110 115 120 Leu Trp Gly Gly Gly Arg Gly Pro Pro Ala Val Leu Ala Ala Val 125 130 135 Pro Ser Pro Pro Pro Ala Ser Pro Arg Ser Gln Tyr Asn Phe Ile 140 145 150 Ala Asp Val Val Glu Lys Thr Ala Pro Ala Val Val Tyr Ile Glu 155 160 165 Ile Leu Asp Arg His Pro Phe Leu Gly Arg Glu Val Pro Ile Ser 170 175 180 Asn Gly Ser Gly Phe Val Val Ala Ala Asp Gly Leu Ile Val Thr 185 190 195 Asn Ala His Val Val Ala Asp Arg Arg Arg Val Arg Val Arg Leu 200 205 210 Leu Ser Gly Asp Thr Tyr Glu Ala Val Val Thr Ala Val Asp Pro 215 220 225 Val Ala Asp Ile Ala Thr Leu Arg Ile Gln Thr Lys Glu Pro Leu 230 235 240 Pro Thr Leu Pro Leu Gly Arg Ser Ala Asp Val Arg Gln Gly Glu 245 250 255 Phe Val Val Ala Met Gly Ser Pro Phe Ala Leu Gln Asn Thr Ile 260 265 270 Thr Ser Gly Ile Val Ser Ser Ala Gln Arg Pro Ala Arg Asp Leu 275 280 285 Gly Leu Pro Gln Thr Asn Val Glu Tyr Ile Gln Thr Asp Ala Ala 290 295 300 Ile Asp Phe Gly Asn Ser Gly Gly Pro Leu Val Asn Leu Asp Gly 305 310 315 Glu Val Ile Gly Val Asn Thr Met Lys Val Thr Ala Gly Ile Ser 320 325 330 Phe Ala Ile Pro Ser Asp Arg Leu Arg Glu Phe Leu His Arg Gly 335 340 345 Glu Lys Lys Asn Ser Ser Ser Gly Ile Ser Gly Ser Gln Arg Arg 350 355 360 Tyr Ile Gly Val Met Met Leu Thr Leu Ser Pro Ser Ile Leu Ala 365 370 375 Glu Leu Gln Leu Arg Glu Pro Ser Phe Pro Asp Val Gln His Gly 380 385 390 Val Leu Ile His Lys Val Ile Leu Gly Ser Pro Ala His Arg Ala 395 400 405 Gly Leu Arg Pro Gly Asp Val Ile Leu Ala Ile Gly Glu Gln Met 410 415 420 Val Gln Asn Ala Glu Asp Val Tyr Glu Ala Val Arg Thr Gln Ser 425 430 435 Gln Leu Ala Val Gln Ile Arg Arg Gly Arg Glu Thr Leu Thr Leu 440 445 450 Tyr Val Thr Pro Glu Val Thr Glu 455 532 amino acids amino acid single linear KIDNFET01 2957969 12 Met Leu Gly Ala Trp Ala Gly Arg Lys Met Ala Asn Val Gly Leu 5 10 15 Gln Phe Gln Ala Ser Ala Gly Asp Ser Asp Pro Gln Ser Arg Pro 20 25 30 Leu Leu Leu Leu Gly Gln Leu His His Leu His Arg Val Pro Trp 35 40 45 Ser His Val Arg Gly Lys Leu Gln Pro Arg Val Thr Glu Glu Leu 50 55 60 Trp Gln Ala Ala Leu Ser Thr Leu Asn Pro Asn Pro Thr Asp Ser 65 70 75 Cys Pro Leu Tyr Leu Asn Tyr Ala Thr Val Ala Ala Leu Pro Cys 80 85 90 Arg Val Ser Arg His Asn Ser Pro Ser Ala Ala His Phe Ile Thr 95 100 105 Arg Leu Val Arg Thr Cys Leu Pro Pro Gly Ala His Arg Cys Ile 110 115 120 Val Met Val Cys Glu Gln Pro Glu Val Phe Ala Ser Ala Cys Ala 125 130 135 Leu Ala Arg Ala Phe Pro Leu Phe Thr His Arg Ser Gly Ala Ser 140 145 150 Arg Arg Leu Glu Lys Lys Thr Val Thr Val Glu Phe Phe Leu Val 155 160 165 Gly Gln Asp Asn Gly Pro Val Glu Val Ser Thr Leu Gln Cys Leu 170 175 180 Ala Asn Ala Thr Asp Gly Val Arg Leu Ala Ala Arg Ile Val Asp 185 190 195 Thr Pro Cys Asn Glu Met Asn Thr Asp Thr Phe Leu Glu Glu Ile 200 205 210 Asn Lys Val Gly Lys Glu Leu Gly Ile Ile Pro Thr Ile Ile Arg 215 220 225 Asp Glu Glu Leu Lys Thr Arg Gly Phe Gly Gly Ile Tyr Gly Val 230 235 240 Gly Lys Ala Ala Leu His Pro Pro Ala Leu Ala Val Leu Ser His 245 250 255 Thr Pro Asp Gly Ala Thr Gln Thr Ile Ala Trp Val Gly Lys Gly 260 265 270 Ile Val Tyr Asp Thr Gly Gly Leu Ser Ile Lys Gly Lys Thr Thr 275 280 285 Met Pro Gly Met Lys Arg Asp Cys Gly Gly Ala Ala Ala Val Leu 290 295 300 Gly Ala Phe Arg Ala Ala Ile Lys Gln Gly Phe Lys Asp Asn Leu 305 310 315 His Ala Val Phe Cys Leu Ala Glu Asn Ser Val Gly Pro Asn Ala 320 325 330 Thr Arg Pro Asp Asp Ile His Leu Leu Tyr Ser Gly Lys Thr Val 335 340 345 Glu Ile Asn Asn Thr Asp Ala Glu Gly Arg Leu Val Leu Ala Asp 350 355 360 Gly Val Ser Tyr Ala Cys Lys Asp Leu Gly Ala Asp Ile Ile Leu 365 370 375 Asp Met Ala Thr Leu Thr Gly Ala Gln Gly Ile Ala Thr Gly Lys 380 385 390 Tyr His Ala Ala Val Leu Thr Asn Ser Ala Glu Trp Glu Ala Ala 395 400 405 Cys Val Lys Ala Gly Arg Lys Cys Gly Asp Leu Val His Pro Leu 410 415 420 Val Tyr Cys Pro Glu Leu His Phe Ser Glu Phe Thr Ser Ala Val 425 430 435 Ala Asp Met Lys Asn Ser Val Ala Asp Arg Asp Asn Ser Pro Ser 440 445 450 Ser Cys Ala Gly Leu Phe Ile Ala Ser His Ile Gly Phe Asp Trp 455 460 465 Pro Gly Val Trp Val His Leu Asp Ile Ala Ala Pro Val His Ala 470 475 480 Gly Glu Arg Ala Thr Gly Phe Gly Val Ala Leu Leu Leu Ala Leu 485 490 495 Phe Gly Arg Ala Ser Glu Asp Pro Leu Leu Asn Leu Val Ser Pro 500 505 510 Leu Gly Cys Glu Val Asp Val Glu Glu Gly Asp Val Gly Arg Asp 515 520 525 Ser Lys Arg Arg Arg Leu Val 530 1542 base pairs nucleic acid single linear BMARNOT02 135360 13 ATATTCTAAA AGGGCACAGT TAATGACGCC TCTTCCTAGT GAATCCGTGT TCTTTATGAG 60 GTATCTTTTA TAGTTGTATC TTTTTTTTTT TCTGAGATGG AGTCTCGCTC TACTGTAGC 120 CAGGATGGAG TGCAGTAGTG TGATCTTGGC TCACTGCAAC CCCTGCCTCC CGGGTTCAA 180 GAATTCTCCT GCCTTAGCCT CCTGAGTAGC TGAGATTACA GGCGCCCACC ACCACACCT 240 GCTGATTTTT GTTTCTTAGT AGAGACAGGG TTTCACCATG TTGGCCAGGC TAGTCTCGA 300 CTGACCTCAA GTGATCCATC CGCCTTGGTC TCCCAAAGTG TTGGGATTAC AGGTGTGAG 360 CACTGTGCCC AGCCAAGTTA TATCTCTAAA GCAATGTGCA AAAATAAACT GAACTTGGG 420 TGATTAGGTA TATTCAACAT TTGTCGGGAG AGTAGATGTT TCATTTTATT TCAGTCCCT 480 TGTAATTTGT CTTCTCTAAT GTTAAATACT ATGTAGAATG TGTCTGTGTA ATTTTATAG 540 TACTTTTATT ATGGATGGAC ATTCTAATTT GTACTGACTT TGGGTCTGTG AACTACTTC 600 ATGTTTGGAG GTTACCAAAA TCTTACCTTT CCCTTTTCTA TTCTAGAATT TACATAGTA 660 ATGACGAAGT TAAGGATAAA GCTTTTGAAC TAGAACTCAG CTGGGTTGGT GAATGTAAG 720 TATTTTTGTA CATTTATTTG CCTTAGGAAT GATCTGTACC ACAGCTAATT TACAACTGA 780 TGTCCTTTCT AATATAATGA AAGCTAAAGC AAATTTACTA GGTTGTCTAA TGAAGGGAA 840 GTTCTGCTTA ATAATTGACT TAAGTTGTGA ACACGTTATT TTTTGAAACA TCCATTTCA 900 GGTTTTAAGA TACTATGCTA TAAATTAATG CTCAGGATTT ATAAATAGCA TAATTTACT 960 TCATTTCCAT AAGAACTTAA TATGTAGGCA CATATAATCT CATGTAGAAG CAGCACAC 1020 AAATATTCGA GTATTACTCA TAGTACAACT TTGCAACCTT AGGTGAGTCA GATATGTG 1080 TTGGGTAGAT CCTATGGTAT ACTGCAAGTT ACAATATGGT ACTCAATTTA AAATTCAT 1140 ACACATGTGG CTTAATTTAC AGTAACTAAT GGAAGACATG AAATTGTTCC AAAAGATA 1200 AGAGAAGAAG CAGAGAAATA TGCTAAGGTA AGCCACAGCA CAAAAACTTC TCTTGGCC 1260 GTACAGTCAG GGAATCTCTT AGCCCAGGAG TTTGAGACCA GCCTGAGCAG CACAGCAA 1320 CCCCCATCCC TAATTTAAAA AAAAAAAAAT TCTCTAACCA AAATTATGTG TTGAATAA 1380 TAAATAGACT GGGGTGGTTT CTATGAAATA ACACTGAGAG TTCAGTTGAA CTAAAGAT 1440 AAATTTTCTA GGTTATCTCT AGTGGGTAAA GTTGCCTTGG TTCCAAAAAA AAAAAACT 1500 GGAGGTTTAG ACTGCAAAGA GTTTTTTAGG ACTTCTAATA CT 1542 3043 base pairs nucleic acid single linear TLYMNOT02 447484 14 CCCACGCGTC CGGTAAATGG CTGTAATACA GGAATTTTGC CACAACCAGT TGGGACAGTC 60 TTGTTGCAAA TACCAGAACC TCAAGAATCG AACAGTGACG CAGGAATAAA TTTAATAGC 120 CTTCCAGCAT TTTCACAGGT GGACCCTGAG GTATTTGCTG CCCTTCCTGC TGAACTTCA 180 AGGGAGCTGA AAGCAGCGTA TGATCAAAGA CAAAGGCAGG GCGAGAACAG CACTCACCA 240 CAGTCAGCCA GCGCATCTGT GCCAAAGAAT CCTTTACTTC ATCTAAAGGC AGCAGTGAA 300 GAAAAGAAAA GAAACAAGAA GAAAAAAACC ATTGGTTCAC CAAAAAGGAT TCAGAGTCC 360 TTGAATAACA AGCTGCTTAA CAGTCCTGCA AAAACTCTGC CAGGGGCCTG TGGCAGTCC 420 CAGAAGTTAA TTGATGGGTT TCTAAAACAT GAAGGACCTC CTGCAGAGAA ACCCCTGGA 480 GAACTCTCTG CTTCTACTTC AGGTGTGCCA GGCCTTTCTA GTTTGCAGTC TGACCCAGC 540 GGCTGTGTGA GACCTCCAGC ACCCAATCTA GCTGGAGCTG TTGAATTCAA TGATGTGAA 600 ACCTTGCTCA GAGAATGGAT AACTACAATT TCAGATCCAA TGGAAGAAGA CATTCTCCA 660 GTTGTGAAAT ACTGTACTGA TCTAATAGAA GAAAAAGATT TGGAAAAACT GGATCTAGT 720 ATAAAATACA TGAAAAGGCT GATGCAGCAA TCGGTGGAAT CGGTTTGGAA TATGGCATT 780 GACTTTATTC TTGACAATGT CCAGGTGGTT TTACAACAAA CTTATGGAAG CACATTAAA 840 GTTACATAAA TATTACCAGA GAGCCTGATG CTCTCTGATA GCTGTGCCAT AAGTGCTTG 900 GAGGTATTTG CAAAGTGCAT GATAGTAATG CTCGGAGTTT TTATAATTTT AAATTTCTT 960 TAAAGCAAGT GTTTTGTACA TTTCTTTTCA AAAAGTGCCA AATTTGTCAG TATTGCAT 1020 AAATAATTGT GTTAATTATT TTACTGTAGC ATAGATTCTA TTTACAAAAT GTTTGTTT 1080 AAAGTTTTAT GGATTTTTAC AGTGAAGTGT TTACAGTTGT TTAATAAAGA ACTGTATG 1140 TATTTTGTAC AGGCTCCTTT TTGTGAATCC TTAAAAACTC AACTCTAGGA AGCAACTA 1200 GTTTATTATA CTAAAAGGCT GAAAAACCTC CAGGCCAGAC TGCTAAGCTC TGAAATTC 1260 GAGAGGTCTC AGACCGGGAT TCTACTTGTT CCAAGAAAGG GTAAAGCTTC TAAACCAT 1320 TATTCTTGTC TCCAAGCATG AACACAGGAG CATGTTAAGA AAATCTTTAC TACTTCTT 1380 ATGCGGAGAA ATCTACATAT TTTGAATTAG AAACACCCTC ACACCCACTT GAAGATTT 1440 TTCCTGGGAA CATTATGTCC CGTAGATCAG AGGTGGTGTT GTCTTTTTGC TTCTACTG 1500 CATTGAGAAA CTTTGATGAT AAAAAAGAAC GGTATAGATT TTTCAAACGT ATATAAAA 1560 TTTTTATGTT ATATGTTATG CCATAACTTT AAAATAAAAA TAGTTTAAAA TTCTATGC 1620 GTGGATATTT GGAACTTTTT CCTCAAACAA ACACCCCACA CTGACTTCAG CAAAACCC 1680 AAACTAGCTA CAGATTACTG CTACGAATGA ATCATTAAGT TTTGTGTCTG CAACAATT 1740 GAAGCACTAA GCCCAAATAT CAGGAAATGT GTGTATGATG GAATTTTCTA GGACAAAA 1800 GATCAAGATT AAAACAGATC AAGATTAATG TATAAAAATG TCTACTAAAA CAGATCAA 1860 TTAAAACAGA TCAAGATTAA TGTATAAAAA TCTCTACTGT TACCAGGTGC TGGCATAC 1920 GGTAGTGTGA TGATAGTTTA GTTTGTAAGA TAATTCTTGT CCTAGGAGGA CAACTTGT 1980 GAGAGAAGCT ACACTAACAT GGAAGCCTAA CAGAGCTTGC TTACTGGTGG ATGTCTGT 2040 TCTTTATTGG TAGTTTGGTT TAGAATTGTG ATGATTACAA TGGACTCGTG ACTACACA 2100 CAGTAAAAAG CAGCCAGCTC TATGGCTATC GGAGGGCAGC TGGAGGGGTC CCCAGCAT 2160 GCAGAGAAAT AAGGTCTGAA GAGCCAGAGG AGTCAGTACC TTTCAGCTGT GACTGGCG 2220 AGGTGGCCGG CGCCATCTCA CCCATCAAGG ACCAGAAAAA CTGCAACTGC TGCTGGGC 2280 TGGCAGCGGC AGGCAACATA GAGACCCTGT GGCGCATCAG TTTCTGGGAT TTTGTGGA 2340 TCTCCGTGCA GGAACTGCTG GACTGTGGCC GCTGTGGGGA TGGCTGCCAC GGTGGCTT 2400 TCTGGGACGC GTTCATAACT GTCCTCAACA ACAGCGGCCT GGCCAGTGAA AAGGACTA 2460 CGTTCCAGGG CAAAGTCAGA GCCCACAGGT GCCACCCCAA GAAGTACCAG AAGGTGGC 2520 GGATCCAGGA CTTCATCATG CTGCAGAACA ACGAGCACAG AATTGCGCAG TACCTGGC 2580 CTTATGGCCC CATCACCGTG ACCATCAACA TGAAGCCCCT TCAGCTATAC CGGAAAGG 2640 TGATCAAGGC CACACCCACC ACCTGTGACC CCCAGCTTGT GGACCACTCT GTCCTGCT 2700 TGGGTTTTGG CAGCGTCAAG TCAGAGGAGG GGATATGGGC AGAGACAGTC TCATCGCA 2760 CTCAGCCTCA GCCTCCACAC CCCACCCCAT ACTGGATCCT GAAGAACTCC TGGGGGGC 2820 AATGGGGAGA GAAGGGCTAT TTCCGGCTGC ACCGAGGGAG CAATACCTGT GGCATCAC 2880 AGTTCCCGCT CACTGCCCGT GTGCAGAAAC CGGATATGAA GCCCCGAGTC TCCTGCCC 2940 CCTGAACCCA CCTGGCCCCC TCAGCTCTGT CCTGTTAGGC CAACTGCCTC CTTGCCAG 3000 CCACCCCCAG GTTTTTGCCC ATCCTCCCAA TCTCAATACA GGG 3043 1081 base pairs nucleic acid single linear PROSTUT03 789927 15 AGGAGGCAGA GGGGGCGTCA GGCCGCGGGA GAGGAGGCCA TGGGCGCGCG CGGGGCGCTG 60 CTGCTGGCGC TGCTGCTGGC TCGGGCTGGA CTCAGGAAGC CGGAGTCGCA GGAGGCGGC 120 CCCTTATCAG GACCATGCGG CCGACGGGTC ATCACGTCGC GCATCGTGGG TGGAGAGGA 180 GCCGAACTCG GGCGTTGGCC GTGGCAGGGG AGCCTGCGCC TGTGGGATTC CCACGTATG 240 GGAGTGAGCC TGCTCAGCCA CCGCTGGGCA CTCACGGCGG CGCACTGCTT TGAAACCTA 300 AGTGACCTTA GTGATCCCTC CGGGTGGATG GTCCAGTTTG GCCAGCTGAC TTCCATGCC 360 TCCTTCTGGA GCCTGCAGGC CTACTACACC CGTTACTTCG TATCGAATAT CTATCTGAG 420 CCTCGCTACC TGGGGAATTC ACCCTATGAC ATTGCCTTGG TGAAGCTGTC TGCACCTGT 480 ACCTACACTA AACACATCCA GCCCATCTGT CTCCAGGCCT CCACATTTGA GTTTGAGAA 540 CGGACAGACT GCTGGGTGAC TGGCTGGGGG TACATCAAAG AGGATGAGGC ACTGCCATC 600 CCCCACACCC TCCAGGAAGT TCAGGTCGCC ATCATAAACA ACTCTATGTG CAACCACCT 660 TTCCTCAAGT ACAGTTTCCG CAAGGACATC TTTGGAGACA TGGTTTGTGC TGGCAATGC 720 CAAGGCGGGA AGGATGCCTG CTTCGGTGAC TCAGGTGGAC CCTTGGCCTG TAACAAGAA 780 GGACTGTGGT ATCAGATTGG AGTCGTGAGC TGGGGAGTGG GCTGTGGTCG GCCCAATCG 840 CCCGGTGTCT ACACCAATAT CAGCCACCAC TTTGAGTGGA TCCAGAAGCT GATGGCCCA 900 AGTGGCATGT CCCAGCCAGA CCCCTCCTGG CCACTACTCT TTTTCCCTCT TCTCTGGGC 960 CTCCCACTCC TGGGGCCGGT CTGAGCCTAC CTGAGCCCAT GCAGCCTGGG GCCACTGC 1020 AGTCAGGCCC TGGTTCTCTT CTGTCTTGTT TGGTAATAAA CACATTCCAG TTGATGCC 1080 C 1081 2061 base pairs nucleic acid single linear LUNGAST01 877617 16 CTTGAGAGCT CTCAAATACT TGGTCATGGA TGAAGCCGAC CGAATACTGA ATATGGATTT 60 TGAGACAGAG GTTGACAAGC CTCGAGATCG GAAAACATTC CTCTTCTCTG CCACCATGA 120 CAAGAAGGTT CAAAAACTTC AGCGAGCAGC TCTGAAGAAT CCTGTGAAAT GTGCCGTTT 180 CTCTAAATAC CAGACAGTTG AAAAATTACA GCAATATTAT ATTTTTATTC CCTCTAAAT 240 CAAGGATACC TACCTGGTTT ATATTCTAAA TGAATTGGCT GGAAACTCCT TTATGATAT 300 CTGCAGCACC TGTAATAATA CCCAGAGAAC AGCTTTGCTA CTGCGAAATC TTGGCTTCA 360 TGCCATCCCC CTCCATGGAC AAATGAGTCA GAGTAAGCGC CTAGGATCCC TTAATAAGT 420 TAAGGCCAAG GCCCGTTCCA TTCTTCTAGC AACTGACGTT GCCAGCCGAG GTTTGGACA 480 ACCTCATGTA GATGTGGTTG TCAACTTTGA CATTCCTACC CATTCCAAGG ATTACATCC 540 TCGAGTAGGT CGAACAGCTA GAGCTGGGCG CTCCGGAAAG GCTATTACTT TTGTCACAC 600 GTATGATGTG GAACTCTTCC AGCGCATAGA ACACTTAATT GGGAAGAAAC TACCAGGTT 660 TCCAACACAG GATGATGAGG TTATGATGCT GACAGAACGC GTCCCCAGCG ATGTCTCCA 720 CACCGCTGCT GCAACCCCTG CTGCTGCTGC TGCCTCTGCT GAATGTGGAG CCTTCCGGG 780 CCACACTGAT CCGCATCCCT CTTCATCGAG TCCAACCTGG ACGCAGGACC CTGAACCTA 840 TGAGGGGATG GAGAGAACCA GCAGAGCTCC CCAAGTTGGG GGCCCCATCC CCTGGGGAC 900 AGCCCATCTT CGTACCTCTC TCGAACTACA GGGATGTGCA GTATTTTGGG GAAATTGGG 960 TGGGAACGCC TCCACAAAAC TTCACTGTTG CCTTTGACAC TGGCTCCTCC AATCTCTG 1020 TCCCGTCCAG GAGATGCCAC TTCTTCAGTG TGCCCTGCTG GTTACACCAC CGATTTGA 1080 CCAAAGCCTC TAGCTCCTTC CAGGCCAATG GGACCAAGTT TGCCATTCAA TATGGAAC 1140 GGCGGGTAGA TGGAATCCTG AGCGAGGACA AGCTGACTAT TGGTGGAATC AAGGGTGC 1200 CAGTGATTTT CGGGGAGGCT CTCTGGGAGC CCAGCCTGGT CTTCGCTTTT GCCCATTT 1260 ATGGGATATT GGGCCTCGGT TTTCCCATTC TGTCTGTGGA AGGAGTTCGG CCCCCGAT 1320 ATGTACTGGT GGAGCAGGGG CTATTGGATA AGCCTGTCTT CTCCTTTTAC CTCAACAG 1380 ACCCTGAAGA GCCTGATGGA GGAGAGCTGG TCCTGGGGGG CTCGGACCCG GCACACTA 1440 TCCCACCCCT CACCTTCGTG CCAGTCACGG TCCCTGCCTA CTGGCAGATC CACATGGA 1500 GTGTGAAGGT GGGCCCAGGG CTGACTCTCT GTGCCAAGGG CTGTGCTGCC ATCCTGGA 1560 CGGGCACGTC CCTCATCACA GGACCCACTG AGGAGATCCG GGCCCTGCAT GCAGCCAT 1620 GGGGAATCCC CTTGCTGGCT GGGGAGTACA TCATCCTGTG CTCGGAAATC CCAAAGCT 1680 CCGCAGTCTC CTTCCTTCTT GGGGGGGTCT GGTTTAACCT CACGGCCCAT GATTACGT 1740 TCCAGACTAC TCGAAATGGC GTCCGCCTCT GCTTGTCCGG TTTCCAGGCC CTGGATGT 1800 CTCCGCCTGC AGGGCCCTTC TGGATCCTCG GTGACGTCTT CTTGGGGACG TATGTGGC 1860 TCTTCGACCG CGGGGACATG AAGAGCAGCG CCCGGGTGGG CCTGGCGCGC GCTCGCAC 1920 GCGGAGCGGA CCTCGGATGG GGAGAGACTG CGCAGGCGCA GTTCCCCGGG TGACGCCC 1980 GTGAAGCGCA TGCGCAGCGG GTGGTCGCGG AGGTCCTGCT ACCCAGTAAA AATCCACT 2040 TTCCATTGAA AAAAAAAAAA A 2061 1186 base pairs nucleic acid single linear KIDNTUT01 999322 17 TAAGCGTCGC CAGACCAGCC TGAGTGGTCT CACAGACGTT GGTCTGCGTG TTTATCTCCT 60 CTCCCCTCCC ACCCCACCCT GAAGCTGGGA ACACTTGGGG CCAGGACCCA TGCTGTCCA 120 ACTGTGGGAC TCCCCTTGGC CAAGGTGACC ACCATATTGG ATTTTGGGGA TCTTGAGCC 180 GTGTCCAGGA TTGTGCCCGT GTTGGGATGA ATAAGCCAAG GCTAAGAGGT CATGAGATT 240 GCCAGGGTCA TGGGAGAGGA TCTGGGCTTG AGCCCTGCTC CCTGACCCCA CTGCCTCCT 300 GTTTGGGAGT TGAGAAGAGC AGGGTGGGTG GGCAGAGAAG AGGTAGGAGG TGCAGGCTG 360 CGCCATCACA GGTGAGAGGG CAGAGGCTCA CCTGATGGGG ACGAGGCTTG AGGTGGGCT 420 AGGCTGGCCC CCACATCACA TCCAGCCCTG GCGAGTGTCC TTCAGGAGGT GGCTGTGCC 480 CTCCTGGACT CGAACATGTG TGAGCTGATG TACCACCTAG GAGAGCCCAG CCTGGCTGG 540 CAGCGCCTCA TCCAGGACGA CATGCTCTGT GCTGGCTCTG TCCAGGGCAA GAAAGACTC 600 TGCCAGGTGA CTGCAGCTCC TGGTCACCCC ATCCAGTTGT GTGGGCCCTT TAGGCTCAC 660 CTGTCCTGGA CTTTCTCCCC ATGTCCCACA CCTCAGGGTC TCCAGAGGGA CCAGAGTCC 720 TGCCTAGCTC CTTGGCCTCA GCAGCTGATT CTCGAAGGCA CTTGGGGCCC AGGTGTCTC 780 CTCAATGCAG ACCTCATGGG GCCCTCCCTC TCTCTCCCCC AGGGTGACTC CGGGGGGCC 840 CTGGTCTGCC CCATCAATGA TACGTGGATC CAGGCCGGCA TTGTGAGCTG GGGATTCGG 900 TGTGCCCGGC CTTTCCGGCC TGGTGTCTAC ACCCAGGTGC TAAGCTACAC AGACTGGAT 960 CAGAGAACCC TGGCTGAATC TCACTCAGGC ATGTCTGGGG CCCGCCCAGG TGCCCCAG 1020 TCCCACTCAG GCACCTCCAG ATCCCACCCA GTGCTGCTGC TTGAGCTGTT GACCGTAT 1080 TTGCTTGGGT CCCTGTGAAC CATGAGCCAT GGAGTCCGGG ATCCCCTTTC TGGTAGGA 1140 GATGGAATCT AATAATAAAA ACTGTAGGTT TTTTATGTGT AAAAAC 1186 2038 base pairs nucleic acid single linear COLNNOT13 1337018 18 GCAGCTTGCT CAGCGGACAA GGATGCTGGG CGTGAGGGAC CAAGGCCTGC CCTGCACTCG 60 GGCCTCCTCC AGCCAGTGCT GACCAGGGAC TTCTGACCTG CTGGCCAGCC AGGACCTGT 120 TGGGGAGGCC CTCCTGCTGC CTTGGGGTGA CAATCTCAGC TCCAGGCTAC AGGGAGACC 180 GGAGGATCAC AGAGCCAGCA TGGATCCTGA CAGTGATCAA CCTCTGAACA GCCTCGATG 240 CAAACCCCTG CGCAAACCCC GTATCCCCAT GGAGACCTTC AGAAAGGTGG GGATCCCCA 300 CATCATAGCA CTACTGAGCC TGGCGAGTAT CATCATTGTG GTTGTCCTCA TCAAGGTGA 360 TCTGGATAAA TACTACTTCC TCTGCGGGCA GCCTCTCCAC TTCATCCCGA GGAAGCAGC 420 GTGTGACGGA GAGCTGGACT GTCCCTTGGG GGAGGACGAG GAGCACTGTG TCAAGAGCT 480 CCCCGAAGGG CCTGCAGTGG CAGTCCGCCT CTCCAAGGAC CGATCCACAC TGCAGGTGC 540 GGACTCGGCC ACAGGGAACT GGTTCTCTGC CTGTTTCGAC AACTTCACAG AAGCTCTCG 600 TGAGACAGCC TGTAGGCAGA TGGGCTACAG CAGCAAACCC ACTTTCAGAG CTGTGGAGA 660 TGGCCCAGAC CAGGATCTGG ATGTTGTTGA AATCACAGAA AACAGCCAGG AGCTTCGCA 720 GCGGAACTCA AGTGGGCCCT GTCTCTCAGG CTCCCTGGTC TCCCTGCACT GTCTTGCCT 780 TGGGGAGAGC CTGAAGACCC CCCGTGTGGT GGGTGGGGAG GAGGCCTCTG TGGATTCTT 840 GCCTTGGCAG GTCAGCATCC AGTACGACAA ACAGCACGTC TGTGGAGGGA GCATCCTGG 900 CCCCCACTGG GTCCTCACGG CAGCCCACTG CTTCAGGAAA CATACCGATG TGTTCAACT 960 GAAGGTGCGG GCAGGCTCAG ACAAACTGGG CAGCTTCCCA TCCCTGGCTG TGGCCAAG 1020 CATCATCATT GAATTCAACC CCATGTACCC CAAAGACAAT GACATCGCCC TCATGAAG 1080 GCAGTTCCCA CTCACTTTCT CAGGCACAGT CAGGCCCATC TGTCTGCCCT TCTTTGAT 1140 GGAGCTCACT CCAGCCACCC CACTCTGGAT CATTGGATGG GGCTTTACGA AGCAGAAT 1200 AGGGAAGATG TCTGACATAC TGCTGCAGGC GTCAGTCCAG GTCATTGACA GCACACGG 1260 CAATGCAGAC GATGCGTACC AGGGGGAAGT CACCGAGAAG ATGATGTGTG CAGGCATC 1320 GGAAGGGGGT GTGGACACCT GCCAGGGTGA CAGTGGTGGG CCCCTGATGT ACCAATCT 1380 CCAGTGGCAT GTGGTGGGCA TCGTTAGCTG GGGCTATGGC TGCGGGGGCC CGAGCACC 1440 AGGAGTATAC ACCAAGGTCT CAGCCTATCT CAACTGGATC TACAATGTCT GGAAGGCT 1500 GCTGTAATGC TGCTGCCCCT TTGCAGTGCT GGGAGCCGCT TCCTTCCTGC CCTGCCCA 1560 TGGGGATCCC CCAAAGTCAG ACACAGAGCA AGAGTCCCCT TGGGTACACC CCTCTGCC 1620 CAGCCTCAGC ATTTCTTGGA GCAGCAAAGG GCCTCAATTC CTATAAGAGA CCCTCGCA 1680 CCAGAGGCGC CCAGAGGAAG TCAGCAGCCC TAGCTCGGCC ACACTTGGTG CTCCCAGC 1740 CCCAGGGAGA GACACAGCCC ACTGAACAAG GTCTCAGGGG TATTGCTAAG CCAAGAAG 1800 ACTTTCCCAC ACTACTGAAT GGAAGCAGGC TGTCTTGTAA AAGCCCAGAT CACTGTGG 1860 TGGAGAGGAG AAGGAAAGGG TCTGCGCCAG CCCTGTCCGT CTTCACCCAT CCCCAAGC 1920 ACTAGAGCAA GAAACCAGTT GTAATATAAA ATGCACTGCC CTACTGTTGG TATGACTA 1980 GTTACCTACT GTTGTCATTG TTATTACAGC TATGGCCACT ATTATTAAAG AGCTGTGA 2038 994 base pairs nucleic acid single linear COLNNOT27 1798496 19 GTGCAGGAGG AGAAGGAGGA GGAGCAGGAG GTGGAGATTC CCAGTTAAAA GGCTCCAGAA 60 TCGTGTACCA GGCAGAGAAC TGAAGTACTG GGGCCTCCTC CACTGGGTCC GAATCAGTA 120 GTGACCCCGC CCCTGGATTC TGGAAGACCT CACCATGGGA CGCCCCCGAC CTCGTGCGG 180 CAAGACGTGG ATGTTCCTGC TCTTGCTGGG GGGAGCCTGG GCAGGACACT CCAGGGCAC 240 GGAGGACAAG GTGCTGGGGG GTCATGAGTG CCAACCCCAT TCGCAGCCTT GGCAGGCGG 300 CTTGTCCCAG GGCCAGCAAC TACTCTGTGG CGGTGTCCTT GTAGGTGGCA ACTGGGTCC 360 TACAGCTGCC CACTGTAAAA AACCGAAATA CACAGTACGC CTGGGAGACC ACAGCCTAC 420 GAATAAAGAT GGCCCAGAGC AAGAAATACC TGTGGTTCAG TCCATCCCAC ACCCCTGCT 480 CAACAGCAGC GATGTGGAGG ACCACAACCA TGATCTGATG CTTCTTCAAC TGCGTGACC 540 GGCATCCCTG GGGTCCAAAG TGAAGCCCAT CAGCCTGGCA GATCATTGCA CCCAGCCTG 600 CCAGAAGTGC ACCGTCTCAG GCTGGGGCAC TGTCACCAGT CCCCGAGAGA ATTTTCCTG 660 CACTCTCAAC TGTGCAGAAG TAAAAATCTT TCCCCAGAAG AAGTGTGAGG ATGCTTACC 720 GGGGCAGATC ACAGATGGCA TGGTCTGTGC AGGCAGCAGC AAAGGGGCTG ACACGTGCC 780 GGGCGATTCT GGAGGCCCCC TGGTGTGTGA TGGTGCACTC CAGGGCATCA CATCCTGGG 840 CTCAGACCCC TGTGGGAGGT CCGACAAACC TGGCGTCTAT ACCAACATCT GCCGCTACC 900 GGACTGGATC AAGAAGATCA TAGGCAGCAA GGGCTGATTC TAGGATAAGC ACTAGATCT 960 CCTTAATAAA CTCACAACTC TCTGAAAAAA AAAA 994 1318 base pairs nucleic acid single linear UTRSNOT08 2082147 20 TCTGAGGCGC GTGCGCGGCC ACCCCAGCCT AGTCCTCTTC TTGGTGCCAC TGGCTAACTA 60 GGTTGAGAAA CCGGCGCCAC AGGCGCANCA CCTGGCCCGG AGCTGGCCCG CTCCTCCCC 120 CCGAGCCGCC CCCAACAACG CGCCCTCTCC CAGTCCTCAC AAAGGGGCCT AGTCCGGCC 180 CCGGCTCTGG CCGTGAGGGA GCGCTGTGGG GGCGCGCTGC CTTCTGCCTG GAAGTGTTG 240 GCAGGTGGTG GGAGAGCGTC AGGCTTGAAC AACATGATTT TAAAGCACGT GTCTGTCTG 300 CGTTTTTTAC TTTTAGGGTT TTGGCCAAAT TGGGCGAGGG CACAAAATAA CCACTTACC 360 CTTCTCACCG AGGAAGAGCG GGAGAAAGGG TATGGCACAG TCACAAGGGT GGGTGAAAA 420 ATACATCAAG GCCTTTTGTA AAGGCTTCTT TGTGGCGGTG CCTGTGGCAG TGACTTTCT 480 GGATCGGGTC GCCTGTGTGG CAAGAGTAGA AGGAGCATCG ATGCAGCCTT CTTTGAATC 540 TGGGGGGAGC CAGTCATCTG ATGTGGTGCT TTTGAACCAC TGGAAAGTGA GGAATTTTG 600 AGTACACCGT GGTGACATTG TATCATTGGT GTCTCCTAAA AACCCAGAAC AGAAGATCA 660 TAAGAGAGTG ATTGCTCTTG AAGGAGATAT TGTCAGAACC ATAGGACACA AAAACCGGT 720 TGTCAAAGTC CCCCGTGGTC ACATCTGGGT TGAAGGTGAT CATCATGGAC ACAGTTTTG 780 CAGTAATTCT TTTGGGCCGG TTTCCCTAGG ACTTCTGCAT GCCCATGCCA CACATATCC 840 GTGGCCCCCA GAGCGCTGGC AGAAATTGGA ATCTGTTCTT CCTCCAGAGC GCTTACCAG 900 ACAGAGAGAA GAGGAATGAC TGCATGAATC TACCTGAGTT GCTGGCATTG GGAGGCCAG 960 TACTGGAAAG GAATGGAAAA AAGAAGCCTC CAAAAGGGAA AAACTTCTGA CAATATGA 1020 CTGTGCGAGA AATATTTACA GCACATTAAA ACGATCTGTA TTATTAAATA AATAATTT 1080 AAATGTTAAA CAGTATTAAA TGGCACCTGA TTTTGTGTTA AATTTTAGTT CCCTGTTG 1140 TAATGCCCCC AAAATATGCA GACCTTTGGG AATATAAAAA TATTGCACCC ACATGTCT 1200 ATGGGGCTGA ATTTCAGATT ATTTGTTACA TATACTTATT ATATTGATTG TTGGGTTT 1260 ATTTTGGTGC TTGCTGCTGA AATAAATTGA AAATTAATAT TCAAAAAAAA AAAAAAAG 1318 2136 base pairs nucleic acid single linear ENDCNOT03 2170967 21 GGCTCTTTTA AATGACCCCA GGCGTCGTGT ATTGAATCCT AGACTCACGT CCGTCTCGCC 60 GGCGCCCGAG CCAGTCCGCG CGCACCGCGT CTGCGTCCCC GAAAGCCCCG CCCGCAAGG 120 CTGCCCTGCC TACCTGGTCT CCGACGTGCT CGTCTGGAGG GCGGTGCGAG GGGCCGAGC 180 GACAAGATGT TCTTGCTGCC TCTTCCGGCT GCGGGGCGAG TAGTCGTCCG ACGTCTGGC 240 GTGAGACGTT TCGGGAGCCG GAGTCTCTCC ACCGCAGACA TGACGAAGGG CCTTGTTTT 300 GGAATCTATT CCAAAGAAAA AGAAGATGAT GTGCCACAGT TCACAAGTGC AGGAGAGAA 360 TTTGATAAAT TGTTAGCTGG AAAGCTGAGA GAGACTTTGA ACATATCTGG ACCACCTCT 420 AAGGCAGGGA AGACTCGAAC CTTTTATGGT CTGCATCAGG ACTTCCCCAG CGTGGTGCT 480 GTTGGCCTCG GCAAAAAGGC AGCTGGAATC GACGAACAGG AAAACTGGCA TGAAGGCAA 540 GAAAACATCA GAGCTGCTGT TGCAGCGGGG TGCAGGCAGA TTCAAGACCT GGAGCTCTC 600 TCTGTGGAGG TGGATCCCTG TGGAGACGCT CAGGCTGCTG CGGAGGGAGC GGTGCTTGG 660 CTCTATGAAT ACGATGACCT AAAGCAAAAA AAGAAGATGG CTGTGTCGGC AAAGCTCTA 720 GGAAGTGGGG ATCAGGAGGC CTGGCAGAAA GGAGTCCTGT TTGCTTCTGG GCAGAACTT 780 GCACGCCAAT TGATGGAGAC GCCAGCCAAT GAGATGACGC CAACCAGATT TGCTGAAAT 840 ATTGAGAAGA ATCTCAAAAG TGCTAGTAGT AAAACCGAGG TCCATATCAG ACCCAAGTC 900 TGGATTGAGG AACAGGCAAT GGGATCATTC CTCAGTGTGG CCAAAGGATC TGACGAGCC 960 CCAGTCTTCT TGGAAATTCA CTACAAAGGC AGCCCCAATG CAAACGAACC ACCCCTGG 1020 TTTGTTGGGA AAGGAATTAC CTTTGACAGT GGTGGTATCT CCATCAAGGC TTCTGCAA 1080 ATGGACCTCA TGAGGGCTGA CATGGGAGGA GCTGCAACTA TATGCTCAGC CATCGTGT 1140 GCTGCAAAGC TTAATTTGCC CATTAATATT ATAGGTCTGG CCCCTCTTTG TGAAAATA 1200 CCCAGCGGCA AGGCCAACAA GCCGGGGGAT GTTGTTAGAG CCAAAAACGG GAAGACCA 1260 CAGGTTGATA ACACTGATGC TGAGGGGAGG CTCATACTGG CTGATGCGCT CTGTTACG 1320 CACACGTTTA ACCCGAAGGT CATCCTCAAT GCCGCCACCT TAACAGGTGC CATGGATG 1380 GCTTTGGGAT CAGGTGCCAC TGGGGTCTTT ACCAATTCAT CCTGGCTCTG GAACAAAC 1440 TTCGAGGCCA GCATTGAAAC AGGGGACCGT GTCTGGAGGA TGCCTCTCTT CGAACATT 1500 ACAAGACAGG TTGTAGATTG CCAGCTTGCT GATGTTAACA ACATTGGAAA ATACAGAT 1560 GCAGGAGCAT GTACAGCTGC AGCATTCCTG AAAGAATTCG TAACTCATCC TAAGTGGG 1620 CATTTAGACA TAGCAGGCGT GATGACCAAC AAAGATGAAG TTCCCTATCT ACGGAAAG 1680 ATGACTGGGA GGCCCACAAG GACTCTCATT GAGTTCTTAC TTCGTTTCAG TCAAGACA 1740 GCTTAGTTCA GATACTCAAA AATGTCTTCA CTCTGTCTTA AATTGGACAG TTGAACTT 1800 AAGGTTTTTG AATAAATGGA TGAAAATCTT TTAACGGAGA CAAAGGATGG TATTTAAA 1860 TGTAGAACAC AATGAAATTT GTATGCCTTG ATTTTTTTTT TCATTTCACA CAAAGATT 1920 TAAAGGTAAA GTTAATATCT TACTTGATAA GGATTTTTAA GATACTCTAT AAATGATT 1980 AATTTTTAGA ACTTCCTAAT CACTTTTCAG AGTATATGTT TTTCATTGAG AAGCAAAA 2040 GTAACTCAGA TTTGTGATGC TAGGAACATG AGCAAACTGA AAATTACTAT GCACTTGT 2100 GAAACAATAA ATGCAACTTG TTGTGAAAAA AAAAAA 2136 1388 base pairs nucleic acid single linear SMCANOT01 2484218 22 GGAAAATGGC GGCGGCGGCG GCGGCGGCTG CAGCTACGAA CGGGACCGGA GGAAGCAGCG 60 GGATGGAGGT GGATGCAGCA GTAGTCCCCA GCGTGATGGC CTGCGGAGTG ACTGGGAGT 120 TTTCCGTCGC TCTCCATCCC CTTGTCATTC TCAACATCTC AGACCACTGG ATCCGCATG 180 GCTCCCAGGA GGGGCGGCCT GTGCAGGTGA TTGGGGCTCT GATTGGCAAG CAGGAGGGC 240 GAAATATCGA GGTGATGAAC TCCTTTGAGC TGCTGTCCCA CACCGTGGAA GAGAAGATT 300 TCATTGACAA GGAATATTAT TACACCAAGG AGGAGCAGTT TAAACAGGTG TTCAAGGAG 360 TGGAGTTTCT GGGTTGGTAT ACCACAGGGG GGCCACCTGA CCCCTCGGAC ATCCACGTC 420 ATAAGCAGGT GTGTGAGATC ATCGAGAGCC CCCTCTTTCT GAAGTTGAAC CCTATGACC 480 AGCACACAGA TCTTCCTGTC AGCGTTTTTG AGTCTGTCAT TGATATAATC AATGGAGAG 540 CCACAATGCT GTTTGCTGAG CTGACCTACA CTCTGGCCAC AGAGGAAGCG GAACGCATT 600 GTGTAGACCA CGTAGCCCGA ATGACAGCAA CAGGCAGTGG AGAGAACTCC ACTGTGGCT 660 AACACCTGAT AGCACAGCAC AGCGCCATCA AGATGCTGCA CAGCCGCGTC AAGCTCATC 720 TGGAGTACGT CAAGGCCTCT GAAGCGGGAG AGGTCCCCTT TAATCATGAG ATCCTGCGG 780 AGGCCTATGC TCTGTGTCAC TGTCTCCCGG TGCTCAGCAC AGACAAGTTC AAGACAGAT 840 TTTATGATCA ATGCAACGAC GTGGGGCTCA TGGCCTACCT CGGCACCATC ACCAAAACG 900 GCAACACCAT GAACCAGTTT GTGAACAAGT TCAATGTCCT CTACGACCGA CAAGGCATC 960 GCAGGAGAAT GCGCGGGCTC TTTTTCTGAT GAGGGTACTT GAAGGGCTGA TGGACAGG 1020 TCAGGCAACT ATCCCAAAGG GGAGGGCACT ACACTTCCTT GAGAGAAACC GCTGTCAT 1080 ATAAAAGGGG AGCAGCCCCT GAGCACCCCT GCTGGTGGCT CTGTCCTCTG TTAGGCAC 1140 CACTGGTTGG TCAACTTGGA TGTTCATCGA GGCTCATTCT GGCCTTGCTC AGAAGCCC 1200 CTGATGCTCT TCAGTGAGGG AGGCACTACC ATTTGAAGTG ACCCCATGTC AGTCACAT 1260 ACTGGTCTTT AGCAAAGTCC AAGGCTGCCT GCTTCCACCT AAGTGGTCTC TGTTCTAC 1320 TTTAATGTCA CCCTCTACAT CATCTTACCT AGCCCACCCA ACCTTATAAA CATGATAA 1380 GACTACTA 1388 2476 base pairs nucleic acid single linear SINIUCT01 2680548 23 CTCGCGTCCT GGGTGCCGCC TCTGAGTAGG GCGGGCGAGG AGGCAGCCAA GGCGGAGCTG 60 ATGGCTGCGC CGAGGGCGGG GCGGGGTGCA GGCTGGAGCC TTCGGGCATG GCGGGCTTT 120 GGGGGCATTC GCTGGGGGAG GAGACCCCGT TTGACCCCTG ACCTCCGGGC CCTGCTGAC 180 TCAGGAACTT CTGACCCCCG GGCCCGAGTG ACTTATGGGA CCCCCAGTCT CTGGGCCCG 240 TTGTCTGTTG GGGTCACTGA ACCCCGAGCA TGCCTGACGT CTGGGACCCC GGGTCCCCG 300 GCACAACTGA CTGCGGTGAC CCCAGATACC AGGACCCGGG AGGCCTCAGA GAACTCTGG 360 ACCCGTTCGC GCGCGTGGCT GGCGGTGGCG CTGGGCGCTG GGGGGGCAGT GCTGTTGTT 420 TTGTGGGGCG GGGGTCGGGG TCCTCCGGCC GTCCTCGCCG CCGTCCCTAG CCCGCCGCC 480 GCTTCTCCCC GGAGTCAGTA CAACTTCATC GCAGATGTGG TGGAGAAGAC AGCACCTGC 540 GTGGTCTATA TCGAGATCCT GGACCGGCAC CCTTTCTTGG GCCGCGAGGT CCCTATCTC 600 AACGGCTCAG GATTCGTGGT GGCTGCCGAT GGGCTCATTG TCACCAACGC CCATGTGGT 660 GCTGATCGGC GCAGAGTCCG TGTGAGACTG CTAAGCGGCG ACACGTATGA GGCCGTGGT 720 ACAGCTGTGG ATCCCGTGGC AGACATCGCA ACGCTGAGGA TTCAGACTAA GGAGCCTCT 780 CCCACGCTGC CTCTGGGACG CTCAGCTGAT GTCCGGCAAG GGGAGTTTGT TGTTGCCAT 840 GGAAGTCCCT TTGCACTGCA GAACACGATC ACATCCGGCA TTGTTAGCTC TGCTCAGCG 900 CCAGCCAGAG ACCTGGGACT CCCCCAAACC AATGTGGAAT ACATTCAAAC TGATGCAGC 960 ATTGATTTTG GAAACTCTGG AGGTCCCCTG GTTAACCTGG ATGGGGAGGT GATTGGAG 1020 AACACCATGA AGGTCACAGC TGGAATCTCC TTTGCCATCC CTTCTGATCG TCTTCGAG 1080 TTTCTGCATC GTGGGGAAAA GAAGAATTCC TCCTCCGGAA TCAGTGGGTC CCAGCGGC 1140 TACATTGGGG TGATGATGCT GACCCTGAGT CCCAGCATCC TTGCTGAACT ACAGCTTC 1200 GAACCAAGCT TTCCCGATGT TCAGCATGGT GTACTCATCC ATAAAGTCAT CCTGGGCT 1260 CCTGCACACC GGGCTGGTCT GCGGCCTGGT GATGTGATTT TGGCCATTGG GGAGCAGA 1320 GTACAAAATG CTGAAGATGT TTATGAAGCT GTTCGAACCC AATCCCAGTT GGCAGTGC 1380 ATCCGGCGGG GACGAGAAAC ACTGACCTTA TATGTGACCC CTGAGGTCAC AGAATGAA 1440 GATCACCAAG AGTATGAGGC TCCTGCTCTG ATTTCCTCCT TGCCTTTCTG GCTGAGGT 1500 TGAGGGCACC GAGACAGAGG GTTAAATGAA CCAGTGGGGG CAGGTCCCTC CAACCACC 1560 CACTGACTCC TGGGCTCTGA AGAATCACAG AAACACTTTT TATATAAAAT AAAATTAT 1620 CTAGCAACAT ATTATAGTAA AAAATGAGGT GGGAGGGCTG GATCTTTTCC CCCACCAA 1680 GGCTAGAGGT AAAGCTGTAT CCCCCTAAAC TTAGGGGAGA TACTGGAGCT GACCATCC 1740 ACCTCCTATT AAAGAAAATG AGCTGCTGCC ATCTTTTGTG GGCAGTTAGT CAGGTGCT 1800 TCTTTGTGGT GTGGTGGGCT CTGGTCTGTT CTGCTCGGTG CTGGGCCTGG GAGCAAAG 1860 TCCCATGCTT GGCTACAGAT ACTGACAGCT GGCCTCTGAA GGAGGGTGAA AACTTCTG 1920 TGACAGTTCC ACATCCATAG TGCATGGTCT GATGAGTGCG GTTGCTGACA TGGGTTTC 1980 GGTAAGCTCC TGAGGTAATG GCAGCCTCAG ACCCCTGCCA TTAGGGGCCA GTGGTGGT 2040 GCAGAGGGCA GTGGCACTTA GATAATCTGG TTGCTGGTCT GGCCAGGGTA GCGTTCAA 2100 CTCCTGTTGG CCTCTTCACT GAAGGCATCA CCAATGTGGC AGTTGTGCAC CCAGATTC 2160 TGTCCATCAT ATTTGCAGTT ACATTTCATT GCATTGTTGG TAAAGTCACT CTCTGCTA 2220 TCAAAGTTTG GGTTGATGAC AACCTGGAGA ATGTAGTTTC CTGGCTTCAC ATCCGTGA 2280 TCAATCCACT GACAGTCAAT GTCATGCCGG TAGAGATCCC AGCAACCCAC AGTGATGC 2340 TGCTCTCCAA AGTTGGCACA CTCATACCGC TTGGAGACAT CCTCCTGACA CTCAGTGT 2400 TCGAGACAGA AACTAGCTTT GTGGCCCTCA GCCACCTTGG TGCCATTTGG GGTGAGGA 2460 TCATAGTGAG TGAAGA 2476 2231 base pairs nucleic acid single linear KIDNFET01 2957969 24 GTTTGAAACA GCTTCACAAG GCTGGTTATG AAGAAGAAAC TCAAAATAAC AGGAGTGGCT 60 TATGGAACTA CATGGAGGTA ACAGAGGAGG GTACCAACCA AAGGCCCTTG AGCAATCAG 120 ATGTTGGGGG CGTGGGCCGG CAGGAAGATG GCGAACGTGG GGCTGCAGTT CCAGGCGAG 180 GCGGGGGACT CGGACCCACA GAGCCGGCCC CTGCTGCTGC TCGGGCAGCT GCACCACCT 240 CACCGCGTGC CCTGGAGCCA CGTCCGCGGG AAGCTGCAGC CCCGGGTCAC CGAGGAGCT 300 TGGCAGGCTG CCCTGAGCAC GCTCAACCCC AACCCCACGG ACAGCTGTCC CCTCTACCT 360 AACTACGCCA CCGTGGCTGC CCTGCCCTGC AGGGTGAGCC GGCACAACAG CCCCTCGGC 420 GCCCACTTCA TCACGCGGCT GGTGCGGACC TGCCTGCCGC CCGGAGCGCA TCGCTGCAT 480 GTGATGGTCT GCGAGCAGCC GGAGGTCTTT GCTTCCGCCT GTGCCCTGGC CCGGGCCTT 540 CCGCTGTTCA CCCACCGCTC AGGTGCCTCT CGGCGCTTGG AGAAGAAGAC GGTCACCGT 600 GAGTTTTTCC TGGTGGGACA AGACAACGGG CCGGTGGAGG TGTCCACATT GCAGTGCTT 660 GCGAATGCCA CAGACGGCGT GCGGCTAGCA GCCCGCATCG TGGACACACC CTGCAATGA 720 ATGAACACCG ACACCTTCCT CGAGGAGATT AACAAAGTTG GAAAGGAGCT GGGGATCAT 780 CCAACCATCA TCCGGGATGA GGAACTGAAG ACGAGAGGAT TTGGAGGAAT CTATGGGGT 840 GGCAAAGCCG CCCTGCATCC CCCAGCCCTG GCCGTCCTCA GCCACACCCC AGATGGAGC 900 ACGCAGACCA TCGCCTGGGT GGGCAAAGGC ATCGTCTATG ACACTGGAGG CCTCAGCAT 960 AAAGGGAAGA CTACCATGCC GGGGATGAAG CGAGACTGCG GGGGTGCTGC GGCCGTCC 1020 GGGGCCTTCA GAGCCGCAAT CAAGCAGGGT TTCAAAGACA ACCTCCACGC TGTGTTCT 1080 TTGGCTGAGA ACTCGGTGGG GCCCAATGCG ACAAGGCCAG ATGACATCCA CCTGCTGT 1140 TCAGGGAAGA CGGTGGAAAT CAACAACACG GATGCCGAGG GCAGGCTGGT GCTGGCAG 1200 GGCGTGTCCT ATGCTTGCAA GGACCTGGGG GCCGACATCA TCCTGGACAT GGCCACCC 1260 ACCGGGGCTC AGGGCATTGC CACAGGGAAG TACCACGCCG CGGTGCTCAC CAACAGCG 1320 GAGTGGGAGG CCGCCTGTGT GAAGGCGGGC AGGAAGTGTG GGGACCTGGT GCACCCGC 1380 GTCTACTGCC CCGAGCTGCA CTTCAGCGAG TTCACCTCAG CTGTGGCGGA CATGAAGA 1440 TCAGTGGCGG ACCGAGACAA CAGCCCCAGC TCCTGTGCTG GCCTCTTCAT CGCCTCAC 1500 ATCGGCTTCG ACTGGCCCGG AGTCTGGGTC CACCTGGACA TTGCTGCACC GGTGCATG 1560 GGTGAGCGAG CCACAGGCTT CGGTGTGGCC CTCCTGCTGG CGCTCTTCGG CCGTGCCT 1620 GAGGACCCTC TGCTGAACCT GGTGTCCCCA CTGGGCTGTG AGGTGGATGT CGAGGAGG 1680 GACGTGGGGA GGGACTCCAA GAGACGCAGG CTTGTGTGAG CCTCCTGCCT CGGCCCTG 1740 AAACGGGGAT CTTTTACCTC ACTTTGCACT GATTAATTTT AAGCAATTGA AAGATTGC 1800 TTCATATGGG TTTTGGTTTG TCTTTCTGGT CGTCAGCGTG GTGGTGGAAA CAGCTGAA 1860 TTTAGGAGAC AGCTTAGGGT TTGGTGCGGG CCACGGGGAG GGGACCGGGA AGCGCTGG 1920 CTTGTTTCTG TTTGTTACTT ACAGGACTGA GACATCTTCT GTAAACTGCT ACCCCTGG 1980 CCTTCTGCAC CCCGGGGTGA GGCCTCCTGC CTGCCTGGTG CCCTGTCCCA GCCCCAGG 2040 CTGTGCAGGG CACCTGCGTG GCTGACAGCC AGGCTCTTAC TCCAGCCGGG GCTGCCAG 2100 CATCCAGCCA GCCCAGCCCT GTGAAAGATG GAGCTGACTT GCTGCAGGGG ACCTGATT 2160 TAGGGCAAGA GAAGTCACAC TCTGGCCTCT CAGAATTCAC TTGAGGTTCA ATTAAATA 2220 GTCACACCGC C 2231 

What is claimed is:
 1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) a polypeptide comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, c) a biologically active fragment of a), and d) an immunogenic fragment of a).
 2. An isolated polypeptide of claim 1, having a sequence of SEQ ID NO:1.
 3. An isolated polypeptide of claim 1, having a sequence of SEQ ID NO:3.
 4. An isolated polynucleotide encoding a polypeptide of claim
 1. 5. An isolated polynucleotide of claim 4, having a sequence of SEQ ID NO:2 or SEQ ID NO:4.
 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim
 5. 7. A cell transformed with a recombinant polynucleotide of claim
 6. 8. A transgenic organism comprising a recombinant polynucleotide of claim
 6. 9. A method for producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
 10. The method of claim 9, wherein the polypeptide has the sequence of SEQ ID NO: 1 or SEQ ID NO:3.
 11. An isolated antibody which specifically binds to a polypeptide of claim
 1. 12. An isolated polynucleotide comprising a sequence selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, c) a polynucleotide having a sequence complementary to a polynucleotide of a), d) a polynucleotide having a sequence complementary to a polynucleotide of b) and e) an RNA equivalent of a)-d).
 13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
 15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.
 16. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
 18. A composition of claim 17, wherein the polypeptide has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:3.
 19. A method for treating a disease or condition associated with decreased expression of functional NHAP, comprising administering to a patient in need of such treatment the composition of claim
 17. 20. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
 21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.
 22. A method for treating a disease or condition associated with decreased expression of functional NHAP, comprising administering to a patient in need of such treatment a composition of claim
 21. 23. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
 24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.
 25. A method for treating a disease or condition associated with overexpression of NHAP, comprising administering to a patient in need of such treatment a composition of claim
 24. 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim
 1. 27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim
 1. 28. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
 29. A method for assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
 30. A diagnostic test for a condition or disease associated with the expression of CORN in a biological sample, the method comprising: a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
 31. The antibody of claim 11, wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. A composition comprising an antibody of claim 11 and an acceptable excipient.
 33. A method of diagnosing a condition or disease associated with the expression of CORN in a subject, comprising administering to said subject an effective amount of the composition of claim
 32. 34. A composition of claim 32, wherein the antibody is labeled.
 35. A method of diagnosing a condition or disease associated with the expression of NHAP in a subject, comprising administering to said subject an effective amount of the composition of claim
 34. 36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 37. An antibody produced by a method of claim
 36. 38. A composition comprising the antibody of claim 37 and a suitable carrier.
 39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 40. A monoclonal antibody produced by a method of claim
 39. 41. A composition comprising the antibody of claim 40 and a suitable carrier.
 42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.
 43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.
 44. A method of detecting a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3in a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 in the sample.
 45. A method of purifying a polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 from a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 46. A microarray wherein at least one element of the microarray is a polynucleotide of claim
 13. 47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising: a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
 48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim
 12. 49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.
 50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.
 51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.
 52. An array of claim 48, which is a microarray.
 53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.
 54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.
 55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate. 